Affinity binding-based system for detecting particulates in a fluid

ABSTRACT

This invention provides methods and apparatus for detecting and quantifying particulate matter suspended in a fluid. Specifically, the invention provides an integrated, affinity-binding based, analytical system comprising a platform for performing an affinity-binding based assay for specifically binding particulates including microbial cells, and a detection device for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. Methods for using the analytical systems of the invention are also provided.

THIS APPLICATION IS A DIV. OF Ser. No. 08/995,056 Dec. 19, 1997 now U.S.Pat. No. 6,143,247 WHICH IS A CIP OF Ser. No. 08/768,990 Dec. 18, 1996.THIS APPLICATION Ser. No. 09/614,834 Jul. 12, 2000 is a divisionalapplication of U.S. Ser. No. 08/995,056, filed Dec. 19, 1997, now U.S.Pat. No. 6,143,247 which CLAIMS BENEFIT OF Ser. No. 60/034,327 Dec. 20,1996 AND CLAIMS BENEFIT OF 60/000,819 Jun. 27, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus for detecting,characterizing and quantifying particulate matter suspended in a fluid.More specifically, the invention provides an integrated,affinity-binding based analytical system for detecting particulates,particularly cells, suspended in a fluid, especially a biological fluid.In particular, the invention provides a platform for performing anaffinity-binding based assay for specifically binding particulatesincluding cells, and a detection means for detecting the particulatesspecifically bound to a defined surface or chamber comprising theplatform. In addition, the invention provides such analytical systems tofacilitate cell accumulation in a specific cell accumulation area orchamber of the platform, allowing particulate counting andcharacterization using the platform, as well as high throughputscreening of test compounds to determine the capacity of the compound toaffect cell viability, metabolism or physiology. Devices formanipulating the platforms of the invention are provided comprisingdetection means operatively arranged relative to the platform, as wellas devices that provide detecting means for manually-manipulatedplatforms. Methods for using the platforms of the invention are alsoprovided.

2. Background of the Related Art

Determining the type, concentration and properties of particulates in afluid is important in a variety of contexts. Dust and dirt particles inwater, oil or other industrial fluids can negatively impact on theperformance and useful lifetime of complex machinery. Pyrogens,including bacterial cells, in pharmaceutical products, or manufacturingfacilities making such products, can compromise the safety andreliability of available drugs. Similarly, cells, particularly bacterialcells, that are themselves disease-causing (such as Salmonella spp.) orthat make toxins (such as botulism toxin) are hazardous, andadvantageously are screened in manufacturing and other settings wherefoodstuffs or other consumables are produced. Finally, mammalian cells,including sperm cells and hematopoietic cells, are usefully analyzed inthe corresponding biological fluids for diagnostic and treatmentmonitoring purposes.

Certain methods and apparatus for detecting biological molecules andcells are known in the prior art.

U.S. Pat. No. 3,615,222 issued Oct. 26, 1971 to Mead discloses aspecific binding method for detecting a component of a biological fluid.

U.S. Pat. No. 3,743,482 issued Jul. 3, 1973 to Eisentraut discloses amethod for determining thyroid function

U.S. Pat. No. 3,907,502 issued Sep. 23, 1975 to Brink discloses a methodfor identifying Bence Jones proteins.

U.S. Pat. No. 4,546,460 issued Oct. 8, 1985 to Ando discloses avideodisc autofocus device.

U.S. Pat. No. 5,009,997 issued Apr. 23, 1991 to Shah et al. disclosestwo-site immunometric sandwich assay.

U.S. Pat. No. 5,091,318 issued Feb. 25, 1992 to Anawis et al. disclosesbinding of allergens to a solid phase.

International Application, Publication No. WO92/07243, published on Apr.30, 1992 in the name of Cellpro, disclose the use of a biologicalparticle separator.

U.S. Pat. No. 5,137,031 issued Aug. 11, 1992 to Guirguis discloses aurine testing apparatus and cell collection.

U.S. Pat. No. 5,278,048 issued Jan. 11, 1994 to Parce et al. disclosesan apparatus for detecting the effect of a test compound on a livingcell.

U.S. Pat. No. 5,296,375, issued Mar. 22, 1994 to Kricka et al. disclosemicroplatforms for detecting the presence of an analyte in a fluid.

U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Kricka et al. disclosemicroplatforms for detecting the presence of an analyte in a fluid.

International Application, Publication No. WO94/16543, published on Jul.21, 1994 in the name of Schutze et al., disclose the use of a laseroptical trap for manipulating living cells.

U.S. Pat. No. 5,338,689 issued Aug. 16, 1994 to Yves et al. discloses amethod for detecting antigens and antibodies.

U.S. Pat. No. 5,403,720 issued Apr. 4, 1995 to Sato et al. discloses amethod for detecting microorganisms.

U.S. Pat. No. 5,427,946, issued Jun. 27, 1995 to Kricka et al. disclosemicroplatforms for detecting the presence of an analyte in a fluid.

U.S. Pat. No. 5,451,504 issued Sep. 19, 1995 to Fitzpatrick et al.discloses a membrane strip for detecting the presence of an analyte in asample.

European Application, Publication No. EP634654, published on Oct. 4,1995 in the name of Ventura disclose an apparatus for measuring purifiedwater quality.

U.S. Pat. No. 5,460,940 issued Oct. 24, 1995 to Yves et al. discloses amethod for detecting antigens and antibodies.

U.S. Pat. No. 5,460,979 issued Oct. 24, 1995 to Levine et al. disclosesan indirect fluorescent assay of blood samples.

U.S. Pat. No. 5,491,067 issued Feb. 13, 1996 to Setcavage et al.discloses an agglutination reaction and separation vessel.

U.S. Pat. No. 5,496,697 issued Mar. 5, 1996 to Parce et aL disclose anapparatus for detecting the effect of test compounds on cells.

U.S. Pat. No. 5,498,392, issued Mar. 12, 1996 to Kricka et al. disclosemicroplatforms for detecting the presence of an analyte in a fluid.

U.S. Pat. No. 5,506,141 issued Apr. 9, 1996 to Weinreb et al. disclosesan apertured cell carrier.

U.S. Pat. No. 5,512,432 issued Apr. 30, 1996 to Lapierre et al.discloses methods for detecting antigens and antibodies.

U.S. Pat. No. 5,547,849 issued Aug. 20, 1996 to Baer et al. discloses anapparatus and method for volumetric capillary cytometry.

U.S. Pat. No. 5,556,764 issued Sep. 17, 1996 to Sizto et al. disclosesan apparatus and method for cell counting and classification.

International Application, Publication No. WO96/12962, published on May2, 1996 in the name of Biocircuits Corp. disclose detection of ananalyte using particles and a specific binding pair in the presence of atransparent surface.

U.S. Pat. No. 5,637,469, issued Jun. 10, 1996 to Kricka et al disclosemicroplatforms for detecting the presence of an analyte in a fluid.

U.S. Pat. No. 5,672,861 issued Sep. 30, 1997 to Fairley et al. disclosesan automatic focusing device for a confocal laser microscope.

However, despite this cited prior art, there remains a need in the artfor methods and apparatus to detect particulates in fluids, particularlycells in biological fluids, rapidly, simply, reliably and moreeconomically than available using the prior art.

SUMMARY OF THE INVENTION

This invention provides an integrated, affinity-binding based,analytical apparatus for detecting particulates, particularly cells,suspended in a fluid, preferably a biological fluid. The inventionprovides a platform for performing an affinity-binding based assay forspecifically binding particulates such as cells, preferably microbialcells, especially bacterial cells, and mammalian cells, especiallyhematopoietic cells, and a detection means for detecting theparticulates specifically bound to a defined surface or chambercomprising the platform. Methods for using the platforms of theinvention are also provided.

In one aspect of the invention is provided an affinity-binding based,analytical apparatus for detecting particulates suspended in a fluid.The apparatus provided by the invention comprises a platform having asurface defining a detection chamber, whereby a specific binding reagentis deposited on the surface of the chamber and specifically binds theparticulate to be detected. In preferred embodiments, the specificbinding reagent is an antibody, a ligand, a lectin, an integrin, anantigen, a receptor, a carbohydrate or an adhesion molecule. Preferably,the surface of the detection chamber is also treated with a blockingcompound that discourages non-specific binding to the surface of thechamber. In another preferred embodiment, the platform is a rotatablestructure, most preferably a disk. In a preferred embodiment of thisaspect of the invention, the disk is a microplatform as disclosed inco-owned and co-pending Ser. No. 08/768,990, filed Dec. 18, 1996,incorporated by reference. The platforms of the invention alsopreferably comprise fluid sample input means, overflow reservoirs, washbuffer reservoirs, and fluid waste receptacles, in fluid connection witheach other as described herein, as well as air displacement vents ororifices, or means for removing the fluid component of a sample appliedthereto.

Means for detecting specifically-bound particles in the surface orchamber are also provided. Preferred embodiments of detecting means area light source, particularly a monochromatic light source, and adetector therefor. In addition, preferred embodiments of the platformsof the invention comprise reservoirs containing detectable labelingreagents and moieties for detecting particulates retained on thedetection chambers of the platforms. In preferred embodiments, saidreagents and moieties comprise stains, preferably histochemical stainsand most preferably vital stains, that specifically bind to theparticulates, most preferably cells, in the detection chambers of theplatforms of the invention. In additional preferred embodiments, saidreagents and moieties comprise immunochemical reagents, preferablyantisera and antibodies and most preferably monoclonal antibodies, thatspecifically bind to the particulates, most preferably cells, in thedetection chambers of the platforms of the invention. In preferredembodiments, said antisera and antibodies are labeled with a detectablelabel. Preferred detectable labels include fluorescent labels andenzymatic moieties capable of converting a substrate to a detectableproduct. In alternative preferred embodiments, the detectable reagentscomprise a first antisera or antibody specific for the particulate to bedetected, most preferably a cell, and a second antisera or antibody thatspecifically recognizes and binds to said first antisera or antibody,and is itself detectably labeled. Preferred detectors includephotodetectors, most preferably photodiodes, avalanche photodiodes,photocells and photomultiplier tubes.

In a second embodiment of this aspect of the invention is provided anapparatus that comprises a platform having a surface defining a cellaccumulation chamber, whereby particulates that are cells, preferablymicrobial cells, especially bacterial cells, and mammalian cells,especially hematopoietic cells, accumulate in the chamber and aredetected therein. Preferably, in certain embodiments, the chamber alsocomprises a filtering means having a pore size that prevents the cellsfrom leaving the chamber when the fluid comprising the sample isreplaced by buffer solutions, detection reagents or other fluid volumes.In other embodiments, the chamber preferably comprises a non-specificcell adhesion coating on the surface thereof that retains the cells inthe chamber. In another preferred embodiments, the surface is treated topermit the cells to attach and multiply in the cell accumulation chamberof the platforms of the invention.

In additional preferred embodiments, the platform is a rotatablestructure, most preferably a disk. In a preferred embodiment of thisaspect of the invention, the disk is a microplatform as disclosed inco-owned and co-pending Ser. No. 08/768,990, filed Dec. 18, 1996,incorporated by reference. The platforms of the invention alsopreferably comprise fluid sample input means, overflow reservoirs, washbuffer reservoirs, fluid waste receptacles, or reservoirs containing anamount of a detectable labeling moiety for labeling the cells retainedin the accumulation chamber, in fluid communication with each other asdescribed herein, or air displacement vents or orifices, or means forremoving the fluid component of a sample applied thereto.

In alternative embodiments, the platforms of the invention are providedto detect, quantitate and characterize the effect(s) of a test compoundon a cell, most preferably on the metabolism, physiology or viability ofthe cell. In such embodiments, platforms are provided with reservoirscontaining a test compound and other components therefor. In suchembodiments, cells retained in the cell accumulation chamber of aplatform of the invention are treated with a test compound contained ina reservoir in fluid communication with the cell accumulation chamber.Said test compound is transferred to the cell accumulation chamber, mostpreferably replacing the fluid sample, for a time and under conditionswherein the test compound can have an effect on the cell. Alternatively,the test compound can comprise a component of the cell accumulationchamber as provided. Detection of cell viability, for example usingvital stains, or cellular physiology or metabolism by detectingmetabolites or other cell products produced in response to the testcompound is achieved using the platforms of the invention, whereinreagents for detecting said effect-associated molecules produced by thecells are introduced into the cell accumulation chamber prior todetection. In these embodiments, detection of the effect-associatedmolecules is achieved using reagents specific for said molecules anddetection means specific for said reagents. Most preferably, theeffect-associated molecules are detected using photodetectable reagentssuch as dyes, most preferably fluorescent dyes, which are contained in areservoir in fluid communication with the cell accumulation chamber anddelivered thereto after treatment of the cells with the test compound.

Means for detecting specifically-bound particles in the surface orchamber are also provided. Preferred embodiments of detecting means area light source, particularly a monochromatic light source, and adetector therefor. In addition, preferred embodiments of the platformsof the invention comprise reservoirs containing detectable labelingreagents and moieties for detecting particulates retained on the cellaccumulation chambers of the platforms. In preferred embodiments, saidreagents and moieties comprise stains, preferably histochemical stainsand most preferably vital stains, that specifically bind to theparticulates, most preferably cells, in the cell accumulation chambersof the platforms of the invention. In additional preferred embodiments,said reagents and moieties comprise immunochemical reagents, preferablyantisera and antibodies and most preferably monoclonal antibodies, thatspecifically bind to the particulates, most preferably cells, in thecell accumulation chambers of the platforms of the invention. Inpreferred embodiments, said antisera and antibodies are labeled with adetectable label. Preferred detectable labels include fluorescent labelsand enzymatic moieties capable of converting a substrate to a detectableproduct. In alternative preferred embodiments, the detectable reagentscomprise a first antisera or antibody specific for the particulate to bedetected, most preferably a cell, and a second antisera or antibody thatspecifically recognizes and binds to said first antisera or antibody,and is itself detectably labeled. Preferred detectors includephotodetectors, most preferably photodiodes, avalanche photodiodes,photocells and photomultiplier tubes.

Additional embodiments of each of these aspects of the invention includeplatforms comprising a multiplicity of the components of the celldetection arrays of the invention as defined herein, thereby providingfor the analysis of multiple aliquots of the same sample or multiplesamples on the same platform.

In a second aspect of the invention, an affinity-binding based,analytical apparatus is provided that is a combination of two elements.The first element is a platform as described herein. In preferredembodiments of these aspects of the invention, the platform is arotatable platform having a means for being rotated about a central axiscomprising a rotational element, preferably a hole for a spindle. Inthese aspects of the invention various components of the platform areconnected to one another by channels, most preferably microchannels asdefined herein. The second element in this aspect of the invention is adevice comprising a holding means for accommodating the platforms of theinvention, most preferably also including detecting means for detectingparticulates and most preferably cells on the platforms of theinvention. In preferred embodiments, the devices of the invention areprovided as a device that comprises rotating means and controlling meansthereof, and components of a detecting means operably positioned todetect binding of particulates on the platform surface and mostpreferably in a detection or cell accumulation chamber of the platform.In these aspects of the invention, fluid displacement through thecomponents of the platforms of the invention is motivated by centripetalforce produced by rotation of the platform about the central axis at aspeed and for a time determined by controlling means comprising thedevice. In a preferred embodiment, the platform and device comprise adisk and player/reader device as disclosed in co-owned and co-pendingU.S. Ser. No. 08/768,990, filed Dec. 18, 1996 and incorporated byreference.

Methods for analyzing a fluid, preferably a biological fluid, to detectparticulates suspended therein using the platforms of the invention arealso provided. In preferred embodiments, the methods provided by theinvention comprise the steps of, first, applying a fluid sample to thesurface of the platform, preferably to a fluid sample input means, andmost preferably wherein said means further comprises means for meteringa specific volume of the fluid into a detection or cell accumulationchamber on the surface of the platform.

In certain embodiments of the methods of the invention a metered amountof the fluid sample applied to the platform is transferred to adetection or cell accumulation chamber. For the purposes of thisinvention, the term “a metered amount” will be understood to mean avolumetric amount of the fluid sample that fills a metering means in thefluid sample input means, wherein volumetric amounts greater than themetered amount are removed from the fluid sample input means into anoverflow chamber or fluid waste receptacle. In preferred embodiments,the metered amount of the fluid sample provided on an inventive platformis from about 10 μL to about 500 μL.

In certain embodiments of the methods of the invention the amount of thefluid sample applied to the platform is transferred to a detectionchamber coated with a specific binding reagent and incubated thereuponfor a time and under conditions wherein specific binding is achievedbetween the particulates comprising the fluid sample and the specificbinding reagent, thereby immobilizing the particulate in the detectionchamber, and removing the fluid sample from the chamber. In preferredembodiments, the cells in the detection chamber are washed with asolution, preferably a buffer and more preferably a buffer comprising acomponent, preferably a salt or detergent, that dissociates particulatesretained in the chamber by non-specific binding unrelated to binding ofthe particulate to the specific binding reagent and does not dissociatebinding of the particulate to the specific binding reagent; said washingsolution is preferably removed from the chamber prior to cell detectionto effect removal of non-specifically retained particulates. Theselective removal of non-specifically bound particulates is accomplishedby precisely controlling the surface shear force exerted on theparticulates by the fluid flowing through the detection chamber.Thereafter the presence, identity and number of particulates in thedetection chamber are detected. In certain embodiments of the methods ofthe invention, a solution containing a reagent for detecting aparticulate in the detection chamber is added to the chamber beforedetection of the particulate is accomplished, most preferably bycontacting the cells retained in the detection chamber with suchreagents. In certain embodiments of this aspect of the methods of theinvention, particulates, preferably cells are stained with a specificdye either prior to application to the platform or after theparticulates are retained in the detection chamber. In additionalpreferred embodiments, the reagent is an antisera or antibody, mostpreferably a monoclonal antibody, linked to a detectable marker such asa fluorescent compound. In other preferred embodiments, the reagent isan antisera or antibody, most preferably a monoclonal antibody, linkedto an enzyme capable of converting a substrate to a detectable product;in such embodiments, the appropriate substrate is also added to theparticulates prior to application to the platform or more preferablyafter the particulates are retained in the detection chamber, in aconcentration, for a time and under conditions whereby the substrate isconverted to the detectable product. In preferred embodiments, theparticulates detected in fluid samples using the methods of theinvention are cells, preferably microbial cells, especially bacterialcells, and mammalian cells, most preferably hematopoietic cells.

In other embodiments of the methods of the invention, the metered amountof the fluid sample applied to the platform is transferred to a cellaccumulation chamber that retains the cells therein upon evacuation ofthe chamber of the fluid sample or replacement of the fluid sample withother fluid components of the platforms of the invention. In theseembodiments, the fluid sample is incubated in the cell accumulationchamber for a time and under conditions wherein cells are retained inthe chamber, after which the fluid sample is removed from the chamber.In preferred embodiments, the cells in the cell accumulation chamber arewashed with a solution, preferably a buffer and more preferably a buffercomprising a component, preferably a salt or detergent, that dissociatesnon-cellular particulates from the chamber but does not remove the cellsfrom the chamber; said washing solution is preferably removed from thechamber prior to cell detection to effect removal of non-cellularparticulates. Thereafter the presence, identity and number of cells inthe accumulation chamber are detected.

In alternative embodiments, the effect of a test compound on a cell,most preferably on the metabolism, physiology or viability of the cell,is determined using the methods of the invention. In such embodiments,cells retained in the cell accumulation chamber of a platform of theinvention are treated with a test compound for a time and underconditions wherein the test compound can have an effect on the cell.Detection of cell viability, for example using vital stains, or cellularphysiology or metabolism by detecting metabolites or other cell productsis achieved using the platforms of the invention, wherein reagents fordetecting said effect-associated molecules produced by the cells areintroduced into the cell accumulation chamber prior to detection. Inthese embodiments, detection of the effect-associated molecules isachieved using reagents specific for said molecules and detection meansspecific for said reagents. Most preferably, the effect-associatedmolecules are detected using photodetectable reagents such as dyes, mostpreferably fluorescent dyes.

In certain embodiments of the methods of the invention, a solutioncontaining a reagent for detecting a particulate in the cellaccumulation chamber is added to the chamber before detection of theparticulate is accomplished, most preferably by contacting the cellsretained in the chamber with such reagents. In certain embodiments ofthis aspect of the methods of the invention, particulates, preferablycells are stained with a specific dye either prior to application to theplatform or after the particulates are retained in the cell accumulationchamber. In additional preferred embodiments, the reagent is an antiseraor antibody, most preferably a monoclonal antibody, linked to adetectable marker such as a fluorescent compound. In other preferredembodiments, the reagent is an antisera or antibody, most preferably amonoclonal antibody, linked to an enzyme capable of converting asubstrate to a detectable product; in such embodiments, the appropriatesubstrate is also added to the particulates prior to application to theplatform or more preferably after the particulates are retained in thecell accumulation chamber, in a concentration, for a time and underconditions whereby the substrate is converted to the detectable product.In preferred embodiments, the particulates detected in fluid samplesusing the methods of the invention are cells, preferably microbialcells, especially bacterial cells, and mammalian cells, most preferablyhematopoietic cells.

Certain preferred embodiments of the apparatus of the invention aredescribed in greater detail in the following sections of thisapplication and in the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of affinity-based platforms of theinvention. FIG. 1A is comprised of a substrate 10 coated with a specificbinding reagent comprising a first member of an affinity binding pair11. FIG. 1B illustrates a substrate 10 coated with a reflective material12, which is coated with a specific binding reagent comprising a firstmember of an affinity binding pair 11. FIG. 1C is a substrate 10, wherethe surface of the substrate is partially coated with a specific bindingreagent comprising a first member of an affinity binding pair 11, andwith a patterned reflective material 12 which is in turn derivatizedwith a blocking agent 13. FIG. 1D is a substrate 14, into which has beenformed pits carrying encoded information. The lower surface of 14 iscoated with a reflective coating 15, and a protective layer 16. Theupper surface of 14 is coated with a specific binding reagent comprisinga first member of an affinity binding pair 11.

FIG. 2 is an exemplar arrangement of platform components useful forenumerating particulates in a fluid, for example cell counting, andcomprises a sample input port 21 connected to an overflow chamber 23 viaa fluid capillary 22. The sample inlet port 21 is fluidly connected tothe binding chamber 24, which is in turn connected to a waste chamber25. Air displacement channels 26 facilitate filling of chambers. Washbuffer chambers 27 and 29, and a dye chamber 28 are fluidicallyconnected to the binding chamber 24 via fluid capillaries 22.

FIG. 3 contains illustrations of arrangements of platform componentsuseful for enumerating particulates in a fluid, for example cellcounting. FIG. 3A comprises a wash buffer chamber 31, linked by acapillary 32 containing a valve 33 to a binding chamber 34. The bindingchamber is linked to a waste chamber 35. FIG. 3B incorporates a dyechamber 36 fluidically connected via a capillary 32 and valve 33 to thebinding chamber 34. FIG. 3C provides an alternate arrangement of thecomponents of FIG. 3B. FIG. 3D adds a sample inlet port 37 and a sampleoverflow chamber 38 to the arrangement of components pictured in FIG.3A. FIG. 3E adds a sample inlet port 37 and a sample overflow chamber 38to the arrangement of components pictured in FIG. 3B. FIG. 3F adds asample inlet port 37 and a sample overflow chamber 38 to the arrangementof components pictured in FIG. 3C. FIG. 3G is similar to FIG. 3C butincorporates a binding chamber 39 containing a number of first membersof the binding pairs. FIG. 3H is similar to 3F except that the bindingchamber 34 has been replaced by a number of chambers in which distinctspecific binding reagents comprising first members of an affinitybinding pair have been deposited.

FIG. 4 contains illustrations of arrangements of platform componentsuseful for studying the effect of a test molecule or molecules onpopulations of cells for enumerating particulates in a fluid, forexample cell counting. FIG. 4A comprises a test molecule chamber 40,linked by a capillary 32 containing a valve 33 to a cell accumulationchamber 43. The binding chamber is linked to a waste chamber 35. FIG. 4Bincorporates a dye chamber 36 fluidically connected via a capillary 32and valve 33 to the binding chamber 34. FIG. 4C provides an alternatearrangement of the components of FIG. 4B. FIG. 4D is similar to FIG. 4C,but with the incorporation of a wash buffer chamber 31 in the fluidicpath between the test molecule chamber 40 and the cell accumulationchamber 43. FIGS. 4E and 4F are is similar to FIG. 4A, with theexception of a multiplicity of test molecule chambers 40 arrayedserially. FIG. 4G is an arrangement in which fluid from a test moleculechamber 40 and a dilution buffer reservoir can be directed to receivingchambers 42 to provide serial dilutions of the test molecule solution.FIG. 4H is another advantageous arrangement of the components of FIG.4G.

FIG. 5 contains schematic illustrations of optical detection systemssuitable for detecting the presence and/or optical properties ofparticulates bound to the platforms of the invention. The apparatus ofFIG. 5A, suitable for transmission, light scattering or directfluorescence measurement of a platform such as illustrated in FIG. 1A,comprises a light source 54, focusing lens system 53, assembly 51comprising optical elements to collect, filter and focus light onto thephotodetector 50. FIG. 5B incorporates the detection elements of FIG.5A, and would be suitable for chemiluminescence or bioluminescencemeasurements. FIG. 5C is a rearrangement of the components of FIG. 5Afor use where the light is reflected from platforms of the type shown inFIG. 1B. FIG. 5D is an apparatus suitable for fluorescence detection onplatforms 1A and 1C, where the assembly of optical elements 55 includeselements such as excitation and emission filters, a dichroic mirror andlenses. FIG. 5E is an apparatus suited for use with platforms 1D. Theelements 56 comprise those necessary to read data from an optical discsuch as a CD-ROM.

FIG. 6 illustrates means and methods for counting and studyingindividual cells using platforms of the type FIG. 1C. FIG. 6Aillustrates an optical apparatus or “head” derived from optical memorystorage and retrieval devices for interrogating a platform 60. Opticalcomponents include diode laser 65, diffraction grating 64, beam splitter63, collimating lenses 62, focusing lens and actuator 61, lens 66, sidelobe detectors 67, and central detector 68. Focusing and tracking areachieved through servo control circuits 69, and actuators 61 and 71. Thesignal derived from interaction of the central beam with the cell 74(e.g. fluorescence emission) is detected by optical detector 68 andamplified by circuitry 70. FIGS. 6B and 6C illustrate advantageousarrangements of reflective tracking features on the platform. In FIG. 6Btrack widths 72 are modulated such that reflections from the two sidelobes 73 produce a modulated signal. Amplitude or frequency modulation,achieved by varying the geometry of the tracks 75, can be used to encodepositional information. FIG. 6C illustrates another arrangement whereinformation is encoded by the presence or absence of reflective featuresin the central track.

FIG. 7 illustrates an apparatus for utilizing tie platforms of theinvention. Optical detection systems 81, such as those illustrated inFIG. 5, are linked to controlling circuitry 82. Output signals aremeasured by the microcontroller 83, which processes the data andcommunicates results through a user interface 84 and/or stores the datain local or nonlocal memory 85 through remote data links. Rotatableplatforms require the use of a motor 87 and motor control circuitry 86,and may also include optical data retrieval and storage means 88.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention provides a platform for performing analytical assays offluid samples, preferably biological fluid samples. For the purposes ofthis invention, the term “sample” will be understood to encompass anyfluid containing a particulate species of interest, wherein theparticulate species is preferably a cell and more preferably microbialcells or somatic cells and most preferably bacterial cells andhematopoietic cells.

For the purposes of this invention, the term “platform” is intended toencompass any solid support structure providing a surface or comprisinga chamber that can be treated to comprise a specific binding reagent.Preferably, the platforms of the invention are rotatable about a centralaxis, providing for movement of sample and other reagents on theplatform under the influence of centripetal force. More preferredembodiments of the platforms of the invention are circular disks thatare rotatable about a central aperture adaptably shaped to accommodate aspindle or other rotating means. Most preferred embodiments of theplatforms of the invention are platforms as disclosed in U.S. PatentApplication U.S. Ser. No. 08/768,990, filed Dec. 18, 1996 andincorporated by reference, and in U.S. Provisional Application, SerialNo. 60/034,327, filed Dec. 20, 1996, the disclosures of each of whichare explicitly incorporated by reference herein.

In one aspect of the platforms of the invention is provided a surface ordetection chamber treated to comprise a specific binding reagent. Forthe purposes of this invention, the term “specific binding reagent” isintended to encompass biomolecules having a specific binding affinitybetween pairs thereof providing a specific molecular binding interactionwith a binding affinity constant of between about 10⁻⁴ and 10⁻¹⁵ M.Examples of such pairs of specific binding reagents include but are notlimited to antigen and antibody, including antisera, polyclonalantibodies and most preferably monoclonal antibodies; receptor andligands, including cell-surface receptors; integrins and adhesionproteins, including ICAM-I and ICAM-II; and carbohydrates and lectins,including phytohemagglutinin. As provided by the invention, specificbinding reagents comprising a first member of a specific binding pair isprovided coating a surface or detection chamber of a platform designedor intended to detect the presence of a particulate, most preferably acell expressing a cognate antigen, receptor or adhesion protein orhaving a carbohydrate moiety at the cell surface specific for aparticular lectin. Said specific binding reagent is applied to thesurface or detection chamber of the platform by depositing the reagenton the surface using any appropriate means, including inkjet printing,computer-positioned syringes, microetching and microlithographicmethods, including photolithography, screen and airbrush printingmethods, solution coating, dipping, and conventional microtitre-welltechniques. In applying said specific binding reagent, the surface ordetection chamber can be treated to provide a two-dimensional array orpattern, wherein certain areas on the surface or detection chamber aretreated with said specific binding reagent and others are not in arecognizable manner. In preferred embodiments, a surface or detectionchamber of the invention is provided having transparent portions coatedwith a specific binding reagent, and other portions coated with areflective material to provide a reflecting surface in a patternalternating with the transparent, coated portions. In alternativeembodiments, a multiplicity of specific binding reagents of distinctspecificity are applied to a surface or detection chamber of theplatform, or each of a multiplicity of specific binding reagents ofdistinct specificity are applied to different areas or regions of asurface or detection chamber of a platform of the invention, therebyproviding a pattern of such distinct specific binding reagents on theplatform. Such arrays can be discrete arrays each comprising a differentspecific binding reagent or can be integrated to comprise a pattern ofeach of the multiplicity of distinct specific binding reagents.Exemplary patterns include alternating strips, checks, and concentriccircles. Similarly, patterns of transparent, specific bindingreagent-coated portions and reflective, non-binding portions areprovided by the invention. Exemplary patterns include alternatingstrips, checks, and concentric circles, most preferably comprising apattern resembling or comprising a “bar code.” In addition, the portionsof the surface of the platform not treated with said specific bindingreagent, and most preferably portions of the surface of the platformcomprising reflective portions are advantageously treated with ablocking agent, such as bovine serum albumin (BSA) to preventnon-specific binding of particulate matter on the surface of theplatform.

Preferred patterns of reflective and specific binding portions of thesurfaces of the platforms of the invention are provided analogous to andas a specific modification of technology developed in the prior art foroptical information storage (e.g. CD-ROM applications). This technologyis based on detecting the presence or absence of signal features havingdimensions on the order of 1 micron embedded in a plastic substrate. Thedetectors or “optical heads” incorporate servomechanisms designed toautomatically position a focal point of light in both a direction normalto the disk and laterally in the plane of the disk to submicronaccuracy. The focusing, tracking and data acquisition functions of theoptical head are implemented using a diode laser, optical elements(diffraction gratings, lenses (including cylindrical lenses), mirrors,polarizers) and multiple detectors. The light emitted from the laser issplit into multiple beams (typically 2 or 3) and directed on to thedisk. The light reflected from each of the beams is coupled back intothe optical head where it is directed to a number of photodiodes(typically 6). The outputs from the photodiodes are combined in variousways (either added or subtracted) to obtain both the data and signalsfor controlling the tracking and focusing. Tracking, or following thedata stream, is accomplished by moving the optical head in a radialdirection, while simultaneously controlling the rate of rotation of thedisk. The radial positioning is derived from the light reflected fromthe regions of the disk on either side of the data pits.

This approach has been modified for detection of particles or cells. Theplatform of FIG. 1D is advantageously provided as an optical diskwherein digital information has been encoded in an standard format;however, in the platforms of the invention, the thickness of thesubstrate is thinned sufficiently so that the presence of particles onthe surface will interfere with the reading of the encoded data usingthe optical detection system pictured in FIG. 5E. A second variationuses the optically transparent substrate is provided having paralleltracks defined by alternating transparent and reflective regions orsurfaces, as is pictured in FIGS. 1C, 6B and 6C. This is done, forexample, using established microlithographic methods to selectively etchvacuum-deposited gold from a glass or quartz substrate. In oneembodiment of the platforms of the invention, a pair of reflectivestripes and intervening transparent region are provided to form a“track”. The transparent region contains “data” in the form of thepresence or absence of cells; this “data” is provided in the practice ofthe methods of the invention upon specific particulate binding to thetransparent region, while the reflective regions provides a means foraccomplishing the tracking and focusing. To confine the cells to thetransparent region, it is advantageous to place the cells on the side ofthe substrate on which the reflective material has been deposited. Thepresence of two chemically different surfaces permits selective chemicalmodification of the transparent and reflective surfaces that promotes(i.e. in the transparent region) or prevents (i.e. in the reflectiveregion) adhesion of the cells to be detected.

An optical head similar, in principle, to that used for opticalinformation storage is used as provided by the methods of the inventionto interrogate the plane to which the cells are adhered through thetransparent substrate. The optical head uses a light source (e.g. adiode laser operating at 650 nm) and optical elements to focus multiplebeams of light on or in the plane of the platform adherent to cells orparticles. The secondary or sub-beams are used for tracking andfocusing, while the primary or main beam illuminates the transparentregion comprising the particles or cells. The presence or absence of acell in the track is detected by a number of means, for example bymodulating the transmission of light through the disk to an opposingdetector, or by fluorescent emission. For example, in the case offluorescence, the light emitted light may be detected by a detector(typically a photomultiplier tube (PMT)) and appropriate filterpositioned either directly opposite the light source, at an obliqueangle to the platform and/or the light source, or as part of an opticalhead which has been designed to collect light emitted back from disk(i.e. at an angle of about 0 degrees).

The platforms of the invention advantageously comprise additionalfluid-handling components attached to the surface or detection chamberor cell accumulation chamber on the platforms. These components can befabricated as described below either integral to the disk or as modulesattached to, placed upon, in contact with or embedded in the disk. Suchcomponents are preferably provided in combinations of related componentsas described in further detail herein that are in fluid communication.For the purposes of this invention, the term “in fluid communication” or“fluidly connected” is intended to define components that are operablyinterconnected to allow fluid flow between components. In preferredembodiments, the platform comprises a rotatable platform, morepreferably a disk, whereby fluid movement on the disk is motivated bycentripetal force upon rotation of the disk.

The platforms of the invention further comprise a sample entry port,preferably comprising metering elements to deliver a volumetric amountof sample fluid to the detection or cell accumulation chamber of theplatform. In these embodiments, the platforms of the invention are alsoprovided with an overflow reservoir for retaining excess fluid appliedto the platform in excess of the amount metered into the detection orcell accumulation chamber, most preferably in fluid communication withthe fluid sample input means wherein excess fluid is transferred to theoverflow reservoir by capillary action. The metering sample port isdesigned to rapidly wick in fluid presented to the opening. The overflowchamber is connected to the entry port to take off any excess fluid notwicked into the capillary bed. The volume of the sample is therebydefined by the number and cross section of the capillaries.

Additional chambers on the platform contain fluids such as a wash bufferand staining solution. The fluid components are in fluid communicationvia narrow bore capillaries of defined cross section, which formcapillary valves. Fluid in the chambers and components of the platformsof the invention will be retained until sufficient driving forceovercomes the surface tension of the fluid. Differing cross sectionsallow fluids to be moved independently by controlling the force applied(e.g. by controlling rotation rate).

In embodiments of the apparatus of the invention comprising theinventive platforms, the invention also comprises a device formanipulating the disks of the invention, wherein the disk is rotatedwithin the device to provide centripetal force to effect fluid flow onthe disk. Accordingly, the device provides means for rotating the diskat a controlled rotational velocity, for stopping and starting diskrotation, and advantageously for changing the direction of rotation ofthe disk. Both electromechanical means and control means, as flirterdescribed herein, are provided as components of the devices of theinvention. User interface means (such as a keypad and a display) arealso provided.

In such embodiments, fluid (including reagents, samples and other liquidcomponents) movement is controlled by centripetal acceleration due torotation of the platform, and by the selective activation of valvescontrolling the communications between the components of the systemscomprising the platform. The magnitude of centripetal accelerationrequired for fluid to flow at a rate and under a pressure appropriatefor a particular system is determined by factors including but notlimited to the effective radius of the platform, the position angle ofthe structures on the platform with respect to the direction of rotationand the speed of rotation of the platform.

In other embodiments, fluid flow is provided on the platforms of theinvention by mechanical means, including but not limited to pumpingusing the creation of air or liquid pressure between the components ofthe platform to effect fluid movement, using pumping means sufficient toachieve fluid movement. These means might include syringe pumps or HPLCpumps. In certain other embodiments of the platforms of the invention,fluid movement is motivated by rapid manual displacement of the platformfollowed by a sharp stop of such displacement; this might be actuated bya spring mechanism or simply a “flick of the wrist”.

It has been shown (Cozens-Roberts et al., 1990, Biophys. J. 58: 107-125)that cells and particles coated with one member of a binding pair have arelatively well-defined value of surface shear, called the criticalshear rate, S_(c), at which the will detach from a surface to which theyare bound. This value is determined by the number and strength of bondsto the surface, as well as the size of the cell in question. The removalof non-specifically (adventitiously) bound particles or cells iseffected by precisely controlling shear rates at value less than S_(c).

Advantageous components of the platforms of the invention include fluidsample input means, including volumetric metering means, channels forfluid flow between components, reagent reservoirs, mixing chambers,optical reading chambers, and most preferably incubation surfaces ordetection chambers comprising a specific binding reagent depositedthereupon, and cell accumulation chambers comprising non-specific celladhesion compounds, filtering means that retain cells in the chamber ortreated surfaces that permit the cells to attach thereto. Alsoadvantageously comprising certain embodiments of the inventive platformsare valves for controlling fluid flow between components, temperaturecontrol elements, separation channels, air outlet ports, sample outletports, mixing means including magnetic, acoustic and preferablymechanical mixers, liquid and dry reagents, and other components asdescribed herein or known to the skilled artisan.

Sample is applied to the detection or cell accumulation chamber of theplatforms of the invention either directly or more preferably bytransfer of a metered amount of a portion of the sample from a fluidsample input means to the chamber, for example, by the selective openingof valves controlling access to the chamber from the fluid sample inputmeans. Said valves include but are not limited to microvalves asdescribed in more detail below including mechanical, electrical andthermal valve mechanisms, as well as capillary microvalves wherein fluidflow is controlled by the relationship between capillary forces andcentripetal forces acting on the fluid. Reagent reservoirs, wash bufferreservoirs, other fluidic components and the contents thereof areconnected to one another and to the detection and cell accumulationchamber through channels, preferably microchannels as defined herein,controlled by such valves. In preferred embodiments, delivery of fluidsthrough such channels is achieved by the coincident rotation of theplatform for a time and at a rotational velocity sufficient to motivatefluid movement between the desired components, and opening of theappropriate valves. The amount of a fluid, or a reagent comprising afluid, delivered to the detection or cell accumulation chamber is thuscontrolled by the speed of rotation and the time during which the valveto the reagent reservoirs is open.

The apparatus of the invention also provides detection systems fordetecting, monitoring, quantitating or analyzing particulatesspecifically retained on the surface of the platform, in a detectionchamber comprising a specific binding reagent or in a cell accumulationchamber as described herein. Detection systems useful in the manufactureand use of the platforms of the invention include, but are not limitedto, fluorescent, chemiluminescent, colorimetric, or scatteringmeasurements. FIG. 5 illustrates optical systems for effecting thesemeasurements. The apparatus of FIG. 5A, suitable for transmission, lightscattering or direct fluorescence measurement of a platform such asillustrated in FIG. 1A, comprises a light source 54, focusing lenssystem 53, assembly 51 comprising optical elements to collect, filterand focus light onto the photodetector 50. FIG. 5B incorporates thedetection elements of FIG. 5A, and would be suitable forchemiluminescence or bioluminescence measurements. FIG. 5C is arearrangement of the components of FIG. 5A for use where the light isreflected from platforms of the type shown in FIG. 1B. FIG. 5D is anapparatus suitable for fluorescence detection on platforms shown inFIGS. 1A and 1C, where the assembly of optical elements 55 includeselements such as excitation and emission filters, a dichroic mirror andlenses. FIG. 5E is an apparatus suited for use with platforms shown inFIG. 1D. The elements 56 comprise those necessary to read data from anoptical disc such as a CD-ROM. electrochemical and radioactivitydetecting means. Optionally, the detection system can be integral to theplatform and can comprise a simple visual detection means such as thedevelopment of a visible color. Alternatively, the detection system cancomprise a component of a device manipulating the platform, preferablycomprising an optical detecting means. Also included in the inventionare devices comprising a light source for illuminating the platform anda magnifying means to facilitate visual inspection (direct orcomputer-aided imaging) of the platform. Non-optical detection systemssuch as electrochemical and radioactivity detecting means may also beused. Embodiments wherein components of the detecting means compriseboth the platform and the device are also encompassed by the invention.

In certain applications it may be desirable to increase the surface areaavailable for specific binding by particles in order to increase thecapacity for binding particles within a certain area of the device. Theuse of porous filter or tortuously-connected microchannels which havebeen treated with one half of the binding pair would provide such a highsurface area.

In operation, such a system would be similar to an affinitychromatography column. Fluid containing particles would be flowedthrough the porous structure. (Pore size must be chosen to avoidclogging by the largest particles present.) In addition to creating alarge surface area available for binding, the particles will be forcednear the binding surfaces as they flow through the pores, increasing theprobability of binding.

An application of such a system would be in detection of cells eitherthrough a direct or indirect means. In this application, cells may bemixed with a buffer containing antibody-linked labels such as goldnanoparticles (−100 nm in size or smaller than a cell) or enzyme-linkedantibodies. The cells are then flowed through the porous structure,binding via a different binding molecule. In the case of gold solparticles, if a sufficient volume is passed through the filter such thatthe total number of cells captured is large, a diffuse reflectancemeasurement can accurately quantitate the amount of gold trapped in thefilter. In the case of an enzymatic assay, an appropriate substrate maybe flowed into the porous structure and the evolved dyespectrophotometrically measured.

A second application of such a system would be as a cell concentrator.Sample may be flowed into the porous structure and appropriate particlesbind to the material. A very large volume of sample may flow thoroughthe structure, such that eventually the number of trapped particles ismany orders of magnitude orders of magnitude greater in concentrationthat in the fluid.

At this point, direct assays may be performed on the particles trappedin the structure. Another possibility is to flow a buffer through thestructure which dissociates the affinity bond between particle andstructure. The highly-concentrated particles may then be transported toanother reservoir for further processing.

In preferred embodiments, the detection or cell accumulation chamber ofthe platform of the invention is constructed so that the height (depth)of the chamber is smaller than the other dimensions of the chamber.Preferably, the height (depth) of the chamber ranges from about 25 μm to1 mm. Most preferably, the chamber has a volume of from about 5 μL toabout 1000 μL, more preferably from about 50 μL to about 500 μL.

Platform Design, Coatings and Composition

Platforms of the invention such as disks and the components comprisingsuch platforms are advantageously provided having a variety ofcomposition and surface coatings appropriate for a particularapplication. Platform composition will be a function of structuralrequirements, manufacturing processes, and reagentcompatibility/chemical resistance properties. Specifically, platformsare provided that are made from inorganic crystalline or amorphousmaterials, e.g. silicon, silica, quartz, inert metals, or from organicmaterials such as plastics, for example, poly(methyl methacrylate)(PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate,polyethylene, polystyrene, polyolefins, polypropylene and metallocene.These may be used with unmodified or modified surfaces as describedbelow. Also provided by the invention are platforms made of compositesor combinations of these materials,for example, platforms manufacturesof a plastic material having embedded therein an optically transparentglass surface comprising for example the detection chamber of theplatform.

The surface properties of these materials may be modified for specificapplications. For example, appropriate surface-modification can eitherencourage or suppress cell and/or protein absorption. Surfacemodification can be achieved by silanization, ion implantation andchemical treatment with inert-gas plasmas (i.e., gases through whichelectrical currents are passed to create ionization). In preferredembodiments, a particular portion of the surface of the platform, mostpreferably comprising a chamber in the platform, is treated with aspecific binding reagent. In preferred embodiments, the specific bindingreagent is a protein (e.g., an antibody, a receptor or adhesionprotein), an antigen or a receptor ligand (e.g., a small molecule suchas a peptide), or a lectin (such as phytohemagglutinin) or acarbohydrate that is recognized by a lectin. Preferably, the surface istreated with such specific binding reagents to form an insoluble anddifficult-to-dissociate bond between the reagent and the surface, tominimize loss of the reagent during subsequent treatment steps (such aswashing). The surface is treated with the reagent to saturate thesurface with the reagent over a defined portion of the surface. Inpreferred embodiments, the treatment of the surface with a specificbinding reagent constitutes a pattern of deposition on the surface thancan be recognized, and most preferably, digitized to provide atwo-dimensional map of the surface for orienting a detecting means.Surfaces treated with a multiplicity of specific binding reagents, mostpreferably in a recognizable pattern of deposition, are within the scopeof the invention disclosed herein.

Also provided by the platforms of the invention are surfaces comprisingdetection and cell accumulation detectors that are porous, i.e.comprising a three-dimensional surface in which specific bindingreagents or particulates can be bound.

In advantageous embodiments, the specific binding reagent is depositedon the surface in optically transparent portions thereof in combinationwith deposition in alternative and adjacent regions of the surface witha reflective material treated to prevent particulate binding thereupon.Reflective material of the appropriate feature size is mostadvantageously prepared using methods and means developed for themanufacture of microelectronic circuits. In a process known as“lift-off”, a negative image of the desired features is produced in aphotoresist material using microlithography. A reflective layercomprising one or more metal layers is deposited by evaporation, afterwhich the photoresist and overlying metal is removed (typically bydissolution of the photoresist layer) leaving the desired patterns onthe surface of the substrate. Alternatively, the portion of the surfacecomprising the specific binding reagent is also reflective but isprepared so that this surface can be distinguished from the adjacentreflective surfaces. In these embodiments, the differential patterns ofoptical transmission and reflection are useful for orienting a lightsource of a detecting means, particularly a monochromatic and mostpreferably a coherent or laser light source, over the appropriateportion of the surface of the platform for detecting the particulatesretained thereupon. In particularly preferred embodiments, the surfacecomprises a pattern of reflective coatings that are used to orient,digitize and quantitate the particulates, most preferably cells,contained within a defined area of the platform comprising the detectionor cell accumulation chamber.

In embodiments of the inventive platforms wherein the reflective metallayer is gold, the surface can be treated with omega-substitutedalkanethiol compounds of general formula HS-(CH₂)_(n)—R (where n is aninteger from 1 to about 50, and R is an alkyl, alkenyl, alkynyl, aryl oralkaryl group, or substituted derivatives thereof), to formself-assembled monolayers (SAM). SAMs formed from poly(ethylene glycol)terminated alkane thiols (e.g., R=(CH₂CH₂O)_(m)CH₃) resist proteinadhesion (see Prime et al., 1993, J Amer. Chem. Soc. 115: 10714-10721)and have been used to block cell adhesion (see Singhvi et al., 1994,Science 264: 696-698). Gold surfaces have been coated with SAMs ofhexadecanethiol (n=15, R=—CH₃) to which laminin was adsorbed to providea substrate suitable for cell attachment and growth (Singhvi et al.,ibid.).

The surface of the platform, particularly the area defining thedetection or cell accumulation chamber, is also advantageously treatedwith a non-specific blocking agent or agents to prevent non-specificbinding of particulates, particularly cells, to the surface of theplatform. The nature and extent to which such treatments are necessarydepends strongly on the nature of the surface. For example, a strongcorrelation has been established between water contact angle and celladsorption, with hydrophilic surfaces showing significantly less celladsorption than hydrophobic surfaces (see Ikada, 1994, Biomaterials 15:725). Silicon, silica, and quartz present an inherently high-energy,hydrophilic surface. Alteration of surface properties is attainedthrough hydroxylation (achieved, for example, by NaOH treatment at hightemperatures) or silanization. Substituted silanes and siloxanes areparticularly appropriate for increasing the hydrophilicity of anotherwise hydrophobic surface. These compounds consist of one or severalreactive head-groups which bond (chemically or through hydrogen-bonding)to a substrate, for example, a core region of alkane (—CH₂O—). Thesecompounds also provide a route for more sophisticated alteration ofsurface properties (such as derivation with functional groups to obtainthe surface properties of interest). A wide variety of suchfunctionalities can be introduced at a surface, including vinyl, phenyl,methylene and methoxy groups, as well as surfaces providing mixedfunctionalities. These functional groups not only change grossproperties like liquid contact angle, but provide sites for preferentialadsorption of molecules, either per se or as a result of fartherconjugation of specific binding reagents such as peptide ligands,antibodies and the like. More preferably, the surface is treated afterdeposition of the specific binding reagent with a non-specific blockingagent, including but not limited to bovine serum albumin and casein.

Plastic-based platforms and disks can also be readily treated to achievethe required surface properties. Inert-gas or reactive-gas plasmas areused to alter surface energies through the formation of surfacecomplexes, for example, hydroxyl-rich surfaces for increasedhydrophilicity, or perfluorinated surfaces for increased hydrophobicity.For example, surface graft polymerization is a technique used to graftpolymers or oligomers with the desired surface properties to a substratepolymer chosen for its bulk processability and manufacturing properties,such as a plastic. Commercial methods for initiating graftpolymerization include gamma radiation, laser radiation, thermal ormechanical processing, photochemical processes, plasma, and wet chemicalprocesses (further discussed in Encyclopedia of Polymer Science andTechnology, 2^(nd) ed., (Supplement), Wiley & Sons: New York, 1989, pp675-689). Chemical modification of polymer surfaces (and appropriatepolymers) includes oxidations (polyethylenes), reductions(fluoropolymers), sulfonations, dehydrohalogenations(dehydrofluorination of poly (vinylidene fluoride)) and hydrolyses.While the chemical nature of the surface is altered through chemicalmodification, mechanical properties, durability and chemical resistanceare primarily a function of the substrate plastic. For example, surfacegrafting of poly(ethylene glycol) (PEG) onto polyethylene yields asurface that is both hydrophilic (unlike polyethylene) and resistant towater (PEG is itself soluble in water, while polyethylene is not).Finally, silanization of organic polymer surfaces can also be performed,providing a wide variety of surface energy/chemistry combinations.

Platform Components

The platforms of the invention are preferably provided with amultiplicity of components, either fabricated directly onto theplatform, or placed on the platform as prefabricated modules. Inaddition to the integral components, certain devices and elements can belocated external to the platform, optimally positioned on a device ofthe invention in relation to the platform, or placed in contact with theplatform either while rotating or when at rest. Components optimallycomprising the platforms of the invention or a controlling device incombination therewith include detection chambers, reservoirs, valvingmechanisms, detectors, sensors, temperature control elements, filters,mixing elements, and control systems.

The invention provides a detection chamber or cell accumulation chamber,or a surface or specialized section of the platform, upon which specificcomponents of a fluid sample, preferably cells and most preferablymicrobial cells, especially bacterial cells, and mammalian cells,especially hematopoietic cells, can be retained. In certain preferredembodiments, cells are retained in the chamber by interaction withspecific binding reagents, including but not limited to ligands,lectins, peptides, proteins, antibodies or fragments thereof derivatizedto be retained within the surface of the platform. In other preferredembodiments, cells are retained in an accumulation chamber bynon-specific binding to adhesion molecules, or physical retention usingfiltering or other means as described below, or by allowing the cells toattach to a substrate that has been specifically surface-modified tofacilitate or promote such attachment. Particulates captured by suchspecific binding can be eluted from the surface of the platform andtransferred to a collection reservoir by treatment withappropriately-chosen ionic strength buffers, using conventional methodsdeveloped for immunological or chromatographic techniques. Morepreferably, particulates, particularly cells and most particularlymicrobial cells, can be specifically retained in the detection or cellaccumulation chamber, surface or specialized section of the platform anddetected using detecting means as described herein. In other preferredembodiments, cellular particulates retained in the detection or cellaccumulation chamber, or on any surface or specialized section of theplatform can be counted, localized on the surface, or characterizedusing the detecting means of the apparatus of the invention. Inpreferred embodiments, cells retained in detection or cell accumulationchambers are monitored for viability, metabolism, the effect of viralinfection or drugs on viability or metabolism, or used for toxicityscreening assays.

In advantageous embodiments, cells are retained in a detection or cellaccumulation chamber on a platform of the invention and incubated in thepresence of a test compound for a time and under conditions whereby thetest compound may have an effect on cell metabolism, physiology orviability. The cells are then detected, and most preferablyquantitatively detected, to determine the effect of the test compound onthe cell. For example, cytotoxicity testing can be performed on a cell,most preferably a mammalian cell, by incubating the test compound withcells retained in the cell accumulation chamber on the platform,followed by a determination of cell viability using visual, microscopicor spectrophotometric detection of the exclusion from the treated cellsof a detectably colored “vital” stain, used according to theunderstanding in the art. Such determinations can be performed in thepresence or absence on the test compound, most preferably in multiplexfashion on the same platform, and a comparison made between the observedcell viability in the presence and absence of the test compound.

In additional embodiments, arrays of specific binding reagents,including but not limited to ligands, lectins, peptides, proteins,antibodies or fragments thereof can be deposited in a detection chamberor on a surface or specialized section of a platform, preferably forminga patterned array that can be detected visually or using the detectingmeans disclosed herein. Also provided are such arrays that are digitizedbased on the signal per unit area detected from the array, so that theposition of a signal with a detection chamber, or on a surface orspecialized section of the platform, indicating a particulate can beidentified and specifically tracked during metabolic, viability,toxicity or other assays. The capacity to collect time-integratedinformation in such assays is also advantageously provided.

The platform may contain reservoirs and chambers for containing fluids,such as the washing buffer used to detach nonspecifically boundparticulates from the surface of the detection chamber, or a solution ofa compound to which cells in the cell accumulation chamber are going tobe exposed. The chambers may be prefilled with liquid components andsealed using valving mechanisms, may be filled with dried reagents whichare resolubilized by the addition of a fluid such as water, or may befilled at the time of use with prepared liquid reagents.

The platforms of the invention also are provided comprising reservoirsand chambers for containing fluids, such as the washing buffer used todetach nonspecifically bound particulates from the surface of thedetection chamber, or a solution of a compound to which cells in thecell accumulation chamber are to be exposed. The chambers may beprefilled with liquid components and sealed using valving mechanisms,may be filled with dried reagents which are resolubilized by theaddition of a fluid such as water, or may be filled at the time of usewith prepared liquid reagents.

In certain embodiments, the platforms of the invention also are providedcomprising fluid waste receptacles and overflow receptacles andreservoirs for holding excess fluid comprising unmetered sample, washbuffers and sample fluid after displaced from the detection and cellaccumulation chambers of the platforms of the invention, or any othersource of excess fluid produced in the practice and use of the platformsof the invention. It will be recognized that in some embodiments, suchexcess fluid will be simply removed from the platforms either by userintervention, or by the placement of an outlet in the platform thatpermits excess fluid to drain therefrom.

The components of the platforms of the invention are in fluidic contractwith one another. In preferred embodiments, fluidic contact is providedby microchannels comprising the surface of the platforms of theinvention. Microchannel sizes are optimally determined by specificapplications and by the amount of delivery rates required for eachparticular embodiment of the platforms and methods of the invention.Microchannel sizes can range from 0.1 μm to a value close to the 1 mmthickness of the platform. Microchannel shapes can be trapezoid,circular or other geometric shapes as required. Microchannels preferablyare embedded in a platform having a thickness of about 0.1 to 100 mm,wherein the cross-sectional dimension of the microchannels across thethickness dimension of the platform is less than 500 μm and from 1 to 90percent of said cross-sectional dimension of the platform.

Valving mechanisms are provided to control of fluid movement andtransfer on the platform. The nature of the valves useful in theplatforms of the invention are essentially identical to the valves andmicrovalves disclosed in co-owned and co-pending U.S. Ser. No.08/768,990, filed Dec. 18, 1996, explicitly incorporated by referenceherein. These valve include mechanical, thermal and capillary valves.

Examples of such microvalves include a piezo activator comprising aglass plate sandwiched between two silicon wafers, as described byNakagawa et al. (1990, Proc. IEEE Workshop of Micro Electro MechanicalSystems, Napa Valley, Calif. pp. 89); a pneumatically-actuatedmicrovalve, as described by Veider et al. (1995, Eurosensors IX, pp.284-286, Stockholm, Sweden, Jun. 25-29); a micromachined gas valve (thatis commercially available; Redwood Microsystems, Menlo Park, Calif.;ICSensors, Milpitas, Calif.); a pressure-balanced microvalve, asdisclosed by Huff et al. (1994, 7^(th) International Conference onSolid-State Sensors and Actuators, pp. 98-101); a polymeric relaxationvalve; and a capillary microvalve. In the latter embodiment, which isbased on the use of rotationally-induced fluid pressure to overcomecapillary forces, it is recognized that fluid flow is dependent on theorientation of the surfaces of the components. Fluids which completelyor partially wet the material of the microchannels, reservoirs,detection chambers, etc. (i.e., the components) of the platforms of theinvention which contain them experience a resistance to flow when movingfrom a component of narrow cross-section to one of larger cross-section,while those fluids which do not wet these materials resist flowing fromcomponents of the platforms of the invention of large cross-section tothose with smaller cross-section. This capillary pressure variesinversely with the sizes of the two components, or combinations thereof,the surface tension of the fluid, and the contact angle of the fluid onthe material of the components. Generally, the details of thecross-sectional shape are not important, but the dependence oncross-sectional dimension results in microchannels of dimension lessthan 500 μm exhibit significant capillary pressure. By varying theintersection shapes, materials and cross-sectional areas of thecomponents of the platform of the invention, “valves” are fashioned thatrequire the application of a particular pressure on the fluid to inducefluid flow. This pressure is applied in the disks of the invention byrotation of the disk (which has been shown above to vary with the squareof the rotational frequency, with the radial position and with theextent of the fluid in the radial direction). By varying capillary valvecross-sectional dimensions as well as the position and extent along theradial direction of the fluid handling components of the platforms ofthe invention, capillary valves are formed to release fluid flow in arotation-dependent manner, using rotation rates of from 100 rpm toseveral thousand rpm. This arrangement allows complex, multistep fluidprocesses to be carried out using a pre-determined, monotonic increasein rotational rate.

Control of the valves of the platforms provided by the invention isachieved either using on-platform controller elements, device-specificcontrollers, or a combination thereof.

Optical detecting means are also provided as components of the apparatusof the invention. The photodetectors of the invention are optimallyprovided to detect optical absorbance/transmittance, fluorescence,light-scattering or other optical signals, which are processed andtranslated into data on the position, number and viability of cells onthe platform. Embodiments of such platforms and devices comprisingscanning arrays are also provided. Also advantageously provided by theinvention are detecting means for tracking and focusing the light sourceon the surface of the platform, and actuating means for positioning thelight source on the surface of the platform.

Detection systems for use on the platforms of the invention includespectroscopic, particularly monochromatic and stroboscopic, andelectrochemical detectors (see, for example, Owicki et al., 1992,Biosensors & Biolelectronics 7: 255). Spectroscopic methods using thesedetectors encompass spectroscopy, particularly ultraviolet and visiblelight absorbance, chemiluminescence, and fluorescence spectroscopy. Thedetection systems utilizing the detectors of the invention arepreferably be external and adjacent to the platform. Generally, thedetection systems of the invention comprise a light source and aphotodetector; in certain embodiments (such as chemiluminescence), onlythe photodetector components are required.

The orientation of these components of the detecting means of theapparatus of the invention will be understood to depend on the nature ofthe detection or cell accumulation chamber and the construction thereof.For example, platforms wherein the detection or cell accumulationchamber comprises an optically transparent surface (completely or inpart, see FIGS. 1A and 1C) will advantageously be used with a devicehaving the light source positioned on one side of the platform and thephotodetector positioned on the other side of the platform. Inalternative arrangements, the photodetector is arranged directly acrossfrom the light source (i.e. at an angle of about 180 degrees, as in FIG.5A) or more advantageously obliquely across the platform from the lightsource (i.e. at an angle of between 90 and 180 degrees). In the latterembodiments, the apparatus may also advantageously include a mirror orother means for deflecting the transmitted light to the photodetectingmeans; however, it will be recognized that such mirrors are not requiredin embodiments provided for fluorescence detection.

In alternative arrangements, wherein the surface of the platform at thedetection or cell accumulation chamber comprises a reflecting surface(FIGS. 1B and 1D), the photodetector is advantageously positioned on thesame side of the platform as the light source (FIG. 5C). In preferredembodiments, the photodetector is provided as an integrated component ofan assembly comprising the light source (FIGS. 5D and 5E), wherein thereflected light is detected along the same axis as the incident light(i.e. at about 0 degrees).

The photodetectors of the invention are optimally provided to detectoptical absorbance/transmittance, fluorescence, light-scattering orother optical signals, which are processed and translated into data onthe position, number and viability of cells on the platform. Embodimentsof such platforms and devices comprising scanning arrays are alsoprovided. Also advantageously provided by the invention are detectingmeans for tracking and focusing the light source on the surface of theplatform, and actuating means for positioning the light source on thesurface of the platform.

Detecting means derived from conventional CD or CD-ROM systems are alsoadvantageously provided. The apparatus 56 in FIG. 5E represents anoptical “head” of a CD (see E. W. Williams, 1994, The CD-ROM and OpticalDisc Recording Systems, Oxford University Press, New York) and is usedwith the platform of FIG. 1D, the substrate of which contains dataencoded in an industry standard format, in the form of pits stamped intothe plastic matrix and coated with a reflective coating (e.g.,aluminum). The platform is constructed so that the aluminized pits arethe focal point of the CD laser. Platform fluidics handling componentsare built within and upon this plastic layer. Particulates bound throughthe first member of the binding pair 11 to the surface of the thinnersubstrate 14 scatter light and interfere with the reading of the data.Those skilled in the art recognize that the thickness and index ofrefraction of optical storage media (e.g. CD-ROM) are such that theeffect of light scattering by particles on the surface of the disk isreduced. In this application, the thickness and composition of thesubstrate, fluid layer and cover are chosen such that particulates boundto the surface interfere with the integrity of the data, and the errorsgenerated in reading the data provide a measure of the number ofparticulates bound. The optical detection apparatus pictured in FIG. 5Dand in more detail in FIG. 6A, is to be used with the platform of FIG.1C which contains reflective features such as those pictured in FIGS. 6Band 6C. Optical elements similar to those used in a conventional CD headare used to generate and focus a main beam and side beams on the surfaceof the platform. The reflection of the side beams off of the reflectivefeatures are used to track the central beam along the transparentregions of the platform, so that particulates bound there may bedetected. Those skilled in the art recognize that optical data retrievalsystems use the reflection of the central beam in a servo-controlledfocusing system. This approach differs in that control of the focusingactuator is based on intensities of the reflections from the side beamsoff of the reflective features. The interaction of a particle with thecentral beam may result in fluorescence, absorption, or scattering,which may be detected by the detector 50 within the “head” or by anotherdetector advantageously placed (not shown). These embodiments thusprovide cell sorting capability, cell tracking and cell viabilityinformation, whereby the status of the cells at each point can bedetected and distinguished from each other cell. This capacity enablesthe platforms and devices of the invention to provide cell-specific dataand tracking information.

Preferred spectroscopic methods include fluorescence, wherebyfluorescence detector systems developed for macroscopic uses are adaptedfor use with the platforms of this invention. For example, an excitationsource such as a laser is focused on an optically-transparent section ofthe disk. Light from any analytically-useful portion of theelectromagnetic spectrum can be coupled with a platform material that isspecifically transparent to light of a particular wavelength, permittingspectral properties of the light to be determined by the dye or otherreagent used to illuminate a particulate retained in an detectionchamber or cell incubation chamber or surface or specialized section ofthe platform interrogated by illumination with light. Alternatively, theselection of light at a particular wavelength can be paired with amaterial having geometries and refractive index properties resulting intotal internal reflection of the illuminating light. This enables eitherdetection of material on the surface of the disk through evanescentlight propagation, or multiple reflections through the sample itself,which increases the path length considerably.

In the practice of this aspect of the invention, the outer surface of awaveguide (a fiber optic, a prism, etc.) is coated with the first memberof a binding molecule pair. Particles which express the second member ofthe pair on their surface(s) are then introduced to the fluid which isin contact with the optical waveguide. A sufficient time is allow topass so that the particles may bind through the binding pair to theouter surface of the waveguide. (Gentle agitation of the fluid may speedup this process.) The waveguide is then washed with buffer. Light istransmitted through the waveguide from a source to a photodetector.Light within the waveguide engages in multiple internal reflection as itpasses along the waveguide. It is well known that there is also anevanescent wave penetrating slightly past the surface of the waveguideand into the surrounding fluid. Particles adsorbed to the surface of thewaveguide will both scatter and absorb light in this evanescent wave.The amount by which the radiation transmitted to the detector isdepressed relative to “clean” waveguides can be used to infer the numberof adsorbed particles.

In one example of fluorescence detection using the platforms of theinvention, light of both the fluorescence excitation wavelength and theemitted wavelength are guided through one face of the device. An angleof from about 90° to about 180° is used to separate the excitation andcollection optical trains, more preferably an angle of about 135° toposition the detector obliquely from both the edge of the platform andthe direction of the light from the light source. It is also possible touse other angles, including 0 degrees, whereby the excitation andemitted light travels collinearly. As long as the source light can bedistinguished from the fluorescence signal, any optical geometry can beused. Optical windows suitable for spectroscopic measurement andtransparent to the wavelengths used are included at appropriatepositions (i.e., in detection chamber or cell accumulation chambers orsurfaces or other specialized sections of the platform). The use of thistype of fluorescence has been disclosed by Haab et al. (1995, Anal.Chem. 67: 3253-3260).

Configurations appropriate for evanescent wave systems can provide forfluorescence to be coupled back into a waveguide on the platform,thereby increasing the efficiency of detection. In these embodiments,the optical component preceding the detector can include a dispersiveelement to permit spectral resolution. Fluorescence excitation can alsobe increased through multiple reflections from surfaces in the devicewhenever noise does not scale with path length in the same way as withsignal.

Absorbance measurements can be used to detect a dye or stain, such as avital stain, or other analyte that changes the intensity of transmittedlight by specifically absorbing energy (direct absorbance) or bychanging the absorbance of another component in the system (indirectabsorbance). Absorbance measurements are preferably used in conjunctionwith enzyme-linked detection of the presence of a particulate within adetection chamber of cell accumulation chamber or a surface orspecialized section of the platform. In preferred embodiments, cellularparticulates are detected by vital staining or other cell-specificstaining (such as the use of dyes specific for certain cell types).Optical path geometry is designed to ensure that the absorbance detectoris focused on a light path receiving the maximum amount of transmittedlight from the illuminated sample. Both the light source and thedetector are advantageously positioned external and adjacent to theplatform; in rotatable embodiments of the platforms of the invention,the light source and the detector can be moved in synchrony with theplatform, or more preferably, the light source illuminates the platformstroboscopically so that absorbance/transmittance is sampled insynchrony with rotation of the platform and cyclical positioning of thedetection or cell accumulation chamber with the solitary position of thelight source/detector pair. The detection chamber or cell accumulationchamber, or a surface or other specialized section of the platform canconstitute a cuvette that is illuminated wherein transmitted lightdetected in a single pass or in multiple passes, particularly when usedwith a stroboscopic light signal that illuminates the detection chamberat a frequency equal to the frequency of rotation or multiples thereof.Alternatively, the detection chamber can be a planar waveguide, whereinthe analyte interacts on the face of the waveguide and light absorbanceis the result of attenuated total internal reflection (i.e., the analytereduces the intensity source light if the analyte is sequestered at thesurface of the chamber, using, for example, specific binding to acompound embedded or attached to the chamber surface; see Dessy, 1989,Anal. Chem. 61: 2191). Although preferred embodiments ofabsorbance/transmittance detector arrays are advantageously used withplatforms of the invention comprising an optically transparent surface(permitting a direct light path through the surface of the platform anpositioning of the light source and detector on opposite sides of theplatform), alternative embodiments wherein the detector is positionedobliquely to the light source, or other embodiments wherein the surfaceof the platform comprises a reflective surface and the detector and thelight source are positioned on the same side of the platform are alsoenvisioned and encompassed in this description of the invention. Boththe light source and the detector are advantageously positioned externaland adjacent to the platform; in rotatable embodiments of the platformsof the invention, the light source and the detector can be moved insynchrony with the platform, or more preferably, the detector output issampled periodically to so that the absorbance/transmittance ismeasured.

Indirect absorbance can be used with the same optical design. Forindirect absorbance measurements, the analyte does not absorb the sourcelight; instead, a drop in absorbance of a secondary material is measuredas the analyte displaces it in the sample chamber. Increasedtransmittance therefore corresponds to analyte concentration. Thisdetection schema is advantageously used in embodiments of the inventionwherein the effect of a test compound on a cell is determined, whereinthe detectable analyte is displaced by a metabolite or other moleculeproduced by the cell in response to the presence of the test compound.

Light scattering and turbidity can also be measured on the platform.Optics are configured as described for absorbance measurements. In thisanalysis, the intensity of the transmitted light is related to theconcentration of the light-scattered particles in a sample.Monochromatic light from a light source, advantageously a laser lightsource, is directed across the cross-sectional area of a detectionchamber or cell accumulation chamber on the platform. Light scattered byparticles in a sample, such as cells, is collected at several anglesover the illuminated portion of the chamber (see Rosenzweig et al.,1994, Anal. Chem. 66: 1771-1776). Data reduction is optimally programmeddirectly into the device based on standards such as appropriately-sizedbeads to relate the signal into interpretable results. Using acalibrated set of such beads, fine discrimination between particles ofdifferent sizes can be obtained.

In alternative arrangements, wherein the surface of the platform at thedetection or cell accumulation chamber comprises a reflecting surface,the photodetector is advantageously positioned on the same side of theplatform as the light source. In preferred embodiments, thephotodetector is provided as an integrated component of the lightsource, wherein the reflected light is detected along the same axis asthe incident light (i.e. at about 0°).

It will be understood that the light source supplied with the devices ofthe invention will also be supplied with focusing means and opticalelements to focally illuminate the platform. It will also be recognizedthat the photodetectors supplied with the devices of the invention willbe supplied with light collecting means, including mirrors and otheroptical elements to direct the transmitted, reflected or fluorescentlight from -the detection or cell accumulation chamber to thephotodetector.

Electric potential measurement can also be used for detecting thepresence of a particulate, especially a cell and preferably a microbialcell, on a surface or in a chamber comprising a specific bindingreagent. In these embodiments of the invention, microelectrodes arefabricated of a noble metal, preferably gold or platinum, usingconventional techniques and are imbued with a biological specificity byimpregnating or coating the electrodes with a specific binding reagent.Electropolymerization of organic films, such as polypyrrole orpolyalanine, comprising said specific binding reagent, orelectrochemical reduction of thiol-terminated specific binding reagentscan be used to fashion such specific electrodes. Detection of thepresence or absence of cellular and other particulates is achieved bymeasuring the electrical impedance between one electrode and a solutioncontact, which can advantageously be positioned in contact with thefluid in the detection chamber comprising the electrodes. Measurementsare made using alternating voltages at frequencies from about 10 Hz to 1MHz. Impedance readings are analyzed by a reading device comprising amicroprocessor to determine detection of the presence or absence ofcells and other particulates.

Temperature control elements are provided to control the temperature ofthe platform during incubation of a fluid containing a particulate, mostadvantageously used to facilitate specific binding between theparticulate and the specific binding reagent. A preferred temperature isroom or ambient temperature, although temperatures above ambient (e.g.,37° C.) and below ambient (e.g., 20° C.) are also preferred. Temperaturecontrol is also important for embodiments of the invention used todetect particulates that are cells; in these embodiments, thetemperature is advantageously kept below 42° C. and above 4° C. toprotect the integrity and viability of the cells. The invention thereforprovides heating elements, including heat lamps, direct laser heaters,Peltier heat pumps, resistive heaters, ultrasonication heaters andmicrowave excitation heaters, and cooling elements, including Peltierdevices and heat sinks, radiative heat fins and other components tofacilitate radiative heat loss. Thermal devices are preferably arrayedto control the temperature of the platform over the area of thedetection or cell accumulation chamber or surface, although arrangementswhereby the platform temperature as a whole is controlled are alsopreferred. Preferably, heating and cooling elements comprise the deviceof the invention, although certain elements, such as radiative heattransfer “fins” and other such components may comprise the platforms ofthe invention. The temperature of any particular area on the platform(preferably, the detection chamber or surface) is monitored by resistivetemperature devices (RTD), thermistors, liquid crystal birefringencesensors or by infrared interrogation using IR-specific detectors, andcan be regulated by feedback control systems.

Filters, sieving structures and other means for selectively retaining orfacilitating passage of particulate matter, including cells, cellaggregates, protein aggregates, or other particulate matter comprisingfluids applied to a platform of the invention are provided. Suchfiltering means include microsieving structures that are fabricateddirectly into a fluid handling structure on a platform (e.g., U.S. Pat.No. 5,304,487; International Application, Publication No. WO93/22053;Wilding et al., 1994, Automat. Analyt. Tech. 40: 43-47) or fabricatedseparately and assembled into the fluid handling structures. The sievingstructures are provided with a range of size exclusion orifices and areoptionally arranged sequentially so as to fractionate a sample basedupon the sizes of the constituent parts of the sample. Preferably, suchsieving structures are provided to permit the introduction of specificparticulates into the detection chamber or surface (such as a cell,preferably a microbial cell) while excluding protein aggregates or othernon-specific particle-like constituents of a fluid which could interferewith detection of the particular particulates of interest. Specificallyincluded in such sieving structures are reagents such as beads andparticularly beads coated with a compound such as an antibody having anaffinity for a contaminant or other substance that would interfere withthe assay to be performed. Also provided in certain embodiments of thecell accumulation chamber of the platforms of the invention arefiltering means having a pore size sufficiently chosen to prevent thecells in the chamber from being lost upon evacuation of the chamber ofthe fluid contents thereof, or during fluid replacement, e.g., by a washbuffer.

Mixing elements, are also advantageously provided as components of theplatforms of the invention. Static mixers can be incorporated into fluidhandling structures of the platform by applying a textured surface tochannels or chambers composing the mixer. Two or more channels can bejoined at a position on the platform and their components mixed togetherby hydrodynamic activity imparted upon them by the textured surface ofthe mixing channel or chamber and, for example, by the action ofcentripetal force imparted by a rotating platform. Fluids can be mixedfor the purposes of preparing serial dilutions of test compounds forsubsequent transfer to the cell accumulation chamber, as is illustratedschematically in FIGS. 4G and 4H. Mixing can also be accomplished byrapidly changing the direction of rotation and by physically agitatingthe platform by systems external thereto.

Also provided as components of the apparatus of the invention aredevices for manipulating the platforms of the invention and to providecomponents for control of the operation of the platform, to providedetection means and data acquisition and storage means.

In embodiments of the invention comprising rotatable disks, the deviceis a disk player/reader that controls the function of the disk. Thisdevice comprises mechanisms, spindles and motors that enable the disk tobe loaded and spun, whereby fluid movement is centripetally-motivated asa consequence (i.e., the centripetal acceleration of the rotating diskcauses the fluid to move through the microchannels and in the otherfluidic components of the platform). In addition, the device providesmeans for a user to operate the disk and access and analyze data,preferably using a keypad and computer display.

Integrated electronic processing systems (generally termed “controllers”herein) that include microprocessors and I/O devices provide thecontrolling elements of the platforms of the invention. Such elementscan be fabricated directly onto a platform, but are more preferably andmost advantageously placed off the platform as a component of the deviceprovided in combination with the platform to comprise the apparatus ofthe invention. In preferred embodiments of the apparatus of theinvention, the controllers can be used to control the rotation drivemotor (both speed, duration and direction), system temperature, optics,data acquisition, analysis and storage, and to monitor the state ofsystems integral to the platform. Examples of advantageous controllingmeans are provided in U.S. Ser. No. 08/768,990, filed Dec. 18, 1996,incorporated by reference herein.

Specific examples of rotational controllers are those using rotationsensors adjacent to the motor itself for determining rotation rate, andmotor controller chips (e.g., Motorola MC33035) for driving directionand speed of such motors. Such sensors and chips are generally used in aclosed-loop configuration, using the sensor data to control rotation ofthe disk to a rotational set-point. Similarly, the rotational data fromthese sensors can be converted from a digital train of pulses into ananalog voltage using frequency-to-voltage conversion chips (e.g., TexasInstruments Model LM2917). In this case, the analog signal then providesfeedback to control an analog voltage set=point corresponding to thedesired rotation rate. Controllers may also use the data encoded in thedisk's data-carrying surface in a manner similar to that used incommercially-available compact disk (CD) players. In these embodiments,the digital data read by the laser is used to control rotation ratethrough a phase-locked loop. The rotation rate information inherent inthe frequency of data bits read may be converted to an analog voltage,as described above.

The controllers can also include communication components that allowaccess to external databases and modems for remote data transfer.Specifically, controllers can be integrated into optical read systems inorder to retrieve information contained on the disk, and to writeinformation generated by the analytic systems on the disk to opticaldata storage sections integral to the disk. In these embodiments it willbe understood that both read and write functions are performed on thesurface of the disk opposite to the surface comprising the componentsdisclosed herein.

Information (i.e., both instructions and data, collectively termed“informatics”) pertaining to the control of any particular analyticsystem on the disk can be stored on the disk itself or externally, mostadvantageously by the microprocessor and/or memory of the device of theinvention, or in a computer connected to the device. The information isused by the controller to control the timing and open/closed state ofmicrovalves on the platform, preferably a disk, to determine optimaldisk rotational velocity, to control heating and cooling elements on thedisk or comprising the device, to monitor detection systems, tointegrate data generated by the disk and to implement logic structuresbased on the data collected.

Such informatics controllers are also advantageously provided in devicesused with embodiments of the platforms of the invention not related torotatable platforms.

In one aspect of the invention is provided a device for manipulating theplatform of the invention and that advantageously accesses and writesinformation or initiate processes on the platform. These include themechanical drive and circuitry for rotation monitoring and control,overall system control, data read/write devices, external detectors andactuators for use with the platform, dedicated data and assay processorsfor processing encoded data and assay data, a central processor unit, auser interface, and means for communicating to the platform, the user,and other devices. Mechanical drive and associated circuits includedevices to control and monitor precisely the rotation rate and angularposition of the platform, and devices to select and mount multiple-disksfrom a cassette, turntable, or other multiple-disk storage unit. Systemcontrol units provide overall device control, either pre-programmed oraccessible to the user-interface. Data read/write devices are providedfor reading encoded information from the platform. The device can alsoinclude external actuators comprising optical magneto-optic, magneticand electrical components to actuate microvalves and initiate processeson the platform, as well as external detectors and sensors or componentsof detectors and sensors that operate in concert with other components,including analytic and diagnostic devices.

Components of the devices comprising the apparatus of the inventioninclude the mechanical drive and circuitry for rotation monitoring andcontrol, overall system control, data read/write devices, externaldetectors and actuators for use with the disk, dedicated data and assayprocessors for processing encoded data and assay data, a centralprocessor unit, a user interface, and means for communicating to thedisk, the user, and other devices. Mechanical drive and associatedcircuits include devices to control and monitor precisely the rotationrate and angular position of the disk, and devices to select and mountmultiple-disks from a cassette, turntable, or other multiple-diskstorage unit. System control units provide overall device control,either pre-programmed or accessible to the user-interface. Disk dataread/write devices are provided for reading encoded information from adisk or other medium. Optimally, write-to-disk capabilities areincluded, permitting a section of the disk to contain analytical datagenerated from assays performed on the disk. This option is notadvantageous in uses of the disk where the disks are contaminated withbiological or other hazards, absent means (such as sterilization) forneutralizing the hazard. The device can also include external actuatorscomprising optical magneto-optic, magnetic and electrical components toactuate microvalves and initiate processes on the disk, as well asexternal detectors and sensors or components of detectors and sensorsthat operate in concert with other components on the disk, includinganalytic and diagnostic devices.

Disk data processors are also advantageously incorporated into thedevices of the invention which enable processing and manipulation ofencoded disk data. These components include software used by the deviceCPU, programmable circuits (such as FPGAs, PLAs) and dedicated chipsets(such as ASICs). Also provided are assay processors for processing dataarising from events and assays performed on the disk and detected byexternal detectors or communicated from on-disk components. The devicealso advantageously comprises a central processing unit or computerwhich will allow processing of disk data and assay results data-analysis(through pre-programming); additionally, conventional computercapabilities (word-processing, graphics production, etc.) can beprovided.

A user interface, including keypads, light-pens, monitors, indicators,flat-panel displays, interface through communications options tohost-devices or peripheral devices, and printers, plotters, and graphicsdevices are provided as components of the platform and devices of theinvention. Communication and telecommunications are provided throughstandard hard-wired interfaces (such as RS-32, IEEE-488M SCSI bus),infra-red and optical communications, short-or long-rangetelecommunications (“cellular” telecommunications radio-frequency), andinternal or external modem for manual or automated telephonecommunications.

Disk information comprises both software written to the disk tofacilitate operation of the assays constructed thereupon, and assay datagenerated during use of the by the user. Disk information includesmaterial written to the disk (as optically encoded data) and informationinherent to the disk (e.g., the current status of a valve, which can beaccessed through magnetic pickup or through the reflective properties ofthe coating material at the valve-position) Data written to the disk mayinclude but is not limited to the audio/video/test and machine formatinformation (e.g., binary, binhex, assembler language). This dataincludes system control data used for initiation of control programs tospin the disk, or perform assays, information on disk configuration,disk identity, uses, analysis protocols and programming, protocolsdescriptions, diagnostic programs and test results, point-of-useinformation, analysis results data, and background information. Acquireddata information can be stored as analog or digital and can be raw data,processed data or a combination of both.

System control data include synchronization data to enable the device tofunction at the correct angular velocity/velocities and accelerationsand data relating to physical parameters of disk. Disk configuration andcompatibility data include data regarding the type of disk(configuration of on-disk devices, valves, and reagent, reaction anddetection chambers) used to determine the applicability of desiredtesting protocols; this data provides a functional identity of the typeof disk and capabilities of the disk. It can be also form part of aninteractive feedback system for checking platform components prior toinitiation of an assay on the disk. Disk identify and serial numbers areprovided encoded on each disk to enable exact identification of a diskby fabrication date, disk type and uses, which data are encoded by themanufacturer, and user information, which is written to the disk by theuser. Also included in disk data is a history of procedures performedwith the disk by the user. Also included in the disk data is a historyof procedures performed with the disk, typically written for bothmachine recognition (i.e., how many and which assays remain unused orready for use), as well as information written by the user.

Advantageous embodiments of such devices and controlling and informationprocessing components thereof are disclosed in U.S. Ser. No. 08/768,990,filed Dec. 18, 1996, incorporated by reference.

The invention also provides platforms comprising a multiplicity ofdetection or cell accumulation chambers and arrays of these componentsin fluid communication with sample input means, reservoirs, wastereceptacles and other components of the invention.

It will be recognized by those with skill in the art that suchembodiments are useful for multiplex assay of a single sample or assaysof multiple samples. Platforms of the invention are provided having,forexample, multiple embodiments of the detection or cell accumulationchambers in fluid communication with one or a multiplicity of sampleentry ports, or one or multiple embodiments of a waste receptacles influid communication Kith the detection or cell accumulation chambers ofthe invention. Also provided are multiplex embodiments of stainingreservoirs provided in the platforms of the invention, so that multiplexstaining of particulates, preferably cells, retained on the platforms ofthe invention, can be achieved by the sequential or simultaneousapplication to the detection or cell accumulation chambers of theplatforms of the invention.

Methods and Uses

The present invention offers a great variety of advantageousapplications and embodiments of the apparatus and methods of theinvention. Certain features will be common to most embodiments, however.These features include sample collection; sample application toplatform, including systems adequacy tests at the time of sampleapplication; a variety of specific assays for detecting particulatescomprising a fluid sample; detection and quantitation of saidparticulates; data collection, processing and analysis; datatransmission and storage to memory; data output to the user (includingprinting and screen display); and viability, metabolic, and toxicityassays performed on the platform.

Fluid samples are collected using means appropriate for the particularsample. Blood, for example, is collected in vacuum tubes in a hospitalor laboratory setting, or using a lancet for home or consumer use. Urinecan be collected into a sterile container and applied to the platformusing conventional liquid-transfer technology. Saliva is preferablyapplied to the disk diluted with a small volume of a solution ofdistilled water, mild detergent and sugar flavoring. This solution canbe provided as a mouthwash/gargle for detecting antigens, biologicalsecretions and microorganisms. Amniotic fluid and cerebrospinal fluidare, of necessity, collected using accepted medical techniques byqualified personnel. Cultured cells are collected using establishedmeans for in vitro passage of cells. Milk is collected simply byobserving appropriate levels of care to avoid contamination.

Fluid samples are optimally loaded onto the platform at a positionproximal to the center of rotation in rotatable embodiments of theinvention, to provide the most extensive path across the surface of theplatform, and to maximize the number, length or arrangement offluid-handling components available to interact with the sample.Multiple samples can be applied to the platform comprising an array ofmultiple sample inlet ports. Devices such as those disclosed in FIGS.13A through 13C of co-owned and co-pending U.S. Ser. No. 08/768,990,filed Dec. 18, 1996 are advantageously used for applying the sample.

Configurations of the Fluidics Apparatus for Certain Applications

An embodiment of a device according to the invention is a portable unitno larger than a portable audio CD player consisting of disk-drive,controllers and selectors for programmable or pre-programmed angularacceleration/deceleration profiles for a limited number of procedures.Such a device is advantageous for on-site testing applications. Forexample, a fluidic sample to be tested is introduced to the disk, whichis inserted into the player and the appropriate program chosen. Analysisresults are stored on the disk, to be later read-out by a largerplayer/reader unit, and/or displayed immediately to the user. Resultscan also be stored as the inherent state of an indicator(positive/negative status of litmus paper in different cuvettes, forexample), with no other data collection or analysis performed by thedevice. This data would be accessed by a larger player/reader or byother means outside the field-work environment. Information about thelocation, time, and other conditions of sample collection are enteredthrough the user interface. Such embodiments are useful for particulatetesting in the field, e.g., testing milk samples in a barnyard setting.

Another embodiment is a stand-alone device with active communicationscapabilities and greater functionality. An exemplary application forsuch a device is as a home blood-assay unit. This device is used by anindividual placing a drop of blood on the disk, inserting the disk, andinitiating the assay, preferably simply by pressing a single button. Oneor more cytometric procedures are then performed. Assay data istransferred to software which performs the requisite analysis, eitheron-disk or within the device. The device can also be permanently ortemporarily attached to the home-telephone line and automaticallytransmit either raw or reduced data to a computer at the centrallocation which is used to analyze the data transmitted, compare the datawith accepted standards and/or previous data from the same patient, makea permanent record as part of a patient's device a confirmation ofreceipt of the data, perhaps the data analysis, and advice orsuggested/recommended course of action (such as contacting thephysician).

A desk-top peripheral/host application station constitutes a device asdescribed above with the ability to accept instructions from and respondto a host computer over one of many advantageous data-protocols. Thesystem is capable of acting as host or can transmit data to peripheralsor other networked devices and workstations. Remote accessing ofpre-programmed functions, function re-programming, and real-time controlcapabilities are also provided.

Yet another embodiment of this application is a centralized or bedsideplayer/reader device with associated software located as a nurses'station in a hospital. As tests are performed on disks, the informationis relayed to a physician by telephone, facsimile or pager viashort-range transceiver. Patient identity can be entered at the time ofsample collection by the use of bar codes and light pens attached to thedevice, providing the advantage of positive patient/sampleidentification.

The device can also be provided having the above-capabilities andfunctionalities and in addition having an interface with an integratedcomputer having high-resolution graphics, image-processing and otherfeatures. The computer provides control of the device for performing thefunctions described above for the peripheral system, while physicalintegration greatly increases data-transmission rates. Additionally, theintegrated system is provided with extensive analysis software andbackground data-bases and information. Disk-storage cassettes ofcarousels are also an advantageous feature of such system. An integratedsystem of this type is useful in a large, analytical laboratory setting.

A self-contained, preferably battery-powered, system is useful forapplications in isolated environments. Examples include devices used inremote or hostile setting, such as air, water and soil testing devicesused in the Arctic for environmental purposes, or for use on thebattlefield for toxic chemical detection.

Applications and Uses

The platforms and devices that make up the fluidics manipulationapparatus of the invention have a wide variety of applications, due tothe flexibility of the design, wherein fluids are motivated on theplatform by centripetal force that arises when the platform is rotated.What follows is a short, representative sample of the types ofapplications encompassed within the scope of the instant invention thatis neither exhaustive or intended to be limiting of all of theembodiments of this invention.

The invention is advantageously used for detecting particulates,preferably cells, more preferably microbial cells and most preferablybacterial cells in biological fluids. A specific use for the platformsof the invention is detection of microbial, particularly bacterialcontamination, of milk. In this embodiment, a platform as disclosedherein or in co-owned and co-pending U.S. Ser. No. 08/768,990,incorporated by reference, is prepared by having a surface adsorbablycoated with monoclonal antibody specific to E. coli., with the remainingsites being blocked with bovine serum albumin (BSA). A milk sample or aplurality of milk samples are introduced onto the disk in a sample port,or more preferably an array of sample ports positioned proximal to thecentral rotating axis of the platform. Alternative embodiments comprisesuch arrays in chambers whereby fluid movement is motivated byalternative means, such as pumping mechanisms; however, centripetalforce motivation is preferred. Control of fluid movement is preferablycontrolled by valve mechanisms as disclosed herein. The samples areintroduced into a detection chamber or surface or a multiplicity thereofcomprising the E. coli monoclonal antibody and incubated for 10 to 60min at a temperature of 20° C., ambient temperature, 37° C. or anyappropriate temperature for binding the antibody to bacterial cells. Themilk fluid is then removed from the detection chamber on the platformpreferably by rotating the platform and opening the necessary valvescontrolling egress from the chamber. The detection chamber isoperatively connected to at least one effluent or waste reservoir forretaining fluid purged from the detection chamber. Non-specific bindingof cells and other particulates is removed from the detection chamber bywashing the chamber once or repeatedly with a buffer solution,preferably containing a salt, competitor or other agent that facilitatesremoval of non-specifically-bound cells. Effluent from these washingsteps is voided to the waste chamber through motivation of the fluid(preferably by centripetal force) through the channel connecting thedetection chamber to the waste reservoir.

Detection of specifically-bound particles, preferably microbial cells,is achieved in a variety of ways as follows. Visual inspection of thereaction chamber can be used to resolve cells, by observation of thechamber by an operator, or alternatively an automated or computer-aidedvision system. Optical methods, including absorbance, fluorescence,chemiluminescence, bioluminescence, and light scattering, can beautomated and performed using a device adapted for this purpose,preferably a device comprising means for rotating the platform toprovide fluid movement on the platform. In preferred embodiments of theapparatus of the invention suitable for detecting anc quantitatingindividual particles, preferably cells, the optical array comprises alaser light source and detection means related to conventional CD andCD-ROM technology. In such embodiments, the laser tracks the area ofeach of the detection chambers on the platform, and records absorbance,transmittance, light scattering and fluorescence. Means for recordingthe output detected by the photodiode, an avalanche photodiode, aphotocell or a photomultiplier tube comprising the detector areadvantageously provided as components of the device.

In the practice of certain of these methods, stains and dyes arepreferably added to the cells or fluids to enhance detection of thecells. Specific examples of such stains and dyes include vital stains,cell-specific stains, fluorescent stains such as rhodamine, specificantibodies linked to fluorescent dyes, or antibodies linked to enzymes(such as horse radish peroxidase (HRP) or alkaline phosphate (AP))capable of catalyzing the conversion of a substrate to aspecifically-detectable product, or other embodiments thereof. In thepractice of these methods, the stains and dyes are applied to the cellsprior to specific binding in the detection chamber, or such stains anddyes are applied to the cells in the wash solution of thereafter oncethe cells have been specifically-bound to the detection chamber. Theplatform advantageously comprises reagent reservoirs containing suchstains and dyes connected to the sample port, the detection chamber, orreservoirs containing washing buffers, for the appropriate introductionto the fluid or surface comprising the cells of interest.

Each of the steps of the bacterial cell detection method disclosedherein are optimized for amount, volume and time of incubation. Specificembodiments are optimized for the platforms, devices and detectionsystems used, and for the particulates to be detected. For example, theinvention provides means for both detecting certain bacterial cells anddetermining whether such cells are Gram positive or Gram negative, usingthe appropriate application of specific staining reagents. Cell numbercan be quantitated either directly, by cell counting, or in relation tothe amount of a specific dye or stain retained (or in the case ofenzyme-linked immunoassay, produced) in the detection chamber.

The invention also provides such cells attached to a specific surfacefor toxicity monitoring, such as metabolic monitoring to determine theefficacy of bioactive drugs or other treatments. Ordered arrays of suchsurface are provided in certain embodiments to facilitate a completedetermination of the purity and sterility of certain biological samples,and for cell cytometric and cytometry applications. Exemplaryarrangements of platform components which can be used to carry out thistype of screening assay are presented in FIG. 4, wherein the detectionchamber is substituted with a cell accumulation chamber as describedherein.

The following Examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.

EXAMPLE 1

Cell Counting, Identification and Monitoring

Apparatus and methods for identifying particular cells or cell types ina biological sample are provided by the invention. A platform asdescribed herein and in co-owned and co-pending U.S. Ser. No.08/768,990, filed Dec. 18, 1996, incorporated by reference, is preparedby having a surface advantageously comprising a detection chamberadsorbably coated with monoclonal antibody specific to E. coli., theremaining sites on the surface comprising the detection chamber beingblocked with bovine serum albumin (BSA). A milk sample is introducedonto the platform via a sample inlet port and brought into contact withthe detection chamber comprising the surface coated with the antibody.The milk is incubated in this chamber for 30 min. The platform is thenrotated to remove unwanted materials from the detection chamber and intoa fluid waste receptacle with which the detection chamber is in fluidcommunication. An amount of a buffer appropriate for washing the chamberis then added to the surface of the chamber from a wash buffer reservoirwith which the detection chamber is in fluid communication andcontaining a washing buffer, said buffer being motivated by centrifugalforce and opening of a microvalve. In a useful embodiment, the washingbuffer comprises an E. coli-specific monoclonal antibody covalentlyattached to an enzyme (such as peroxidase). This incubation is allowedto proceed for 5 min. The platform is again rotated with the opening ofthe appropriate microvalves to remove the washing solution from thechamber and to add a solution containing an enzymatic substrate(tetramethylbenzidine and hydrogen peroxide), maintained heretofore in areagent reservoir with which the detection chamber is in fluidcommunication, preferably by a microvalve-controlled microchannel. Theamount of E. coli bound in the reaction chamber is quantitated withregard to the amount of detected enzymatic activity, which is determinedspectrophotometrically by the appearance of a light-absorbing product orthe disappearance of a light-absorbing substrate.

EXAMPLE 2 Somatic Cell Counting

An example of an assay for a mammalian cell in a biological fluid is thedetection of somatic cell in a sample of cow's milk. The assay system isillustrated in FIG. 2 and consists of a sample entry port 21, washbuffer chambers 27 and 29 (containing a wash buffer solution of 25 mMpotassium hydrogen (KH) phthalate, pH 5/0.001% Triton X-100), a dyechamber 28 (containing a staining solution of 0.002% ethidium bromide inKH-phthalate buffer), and a binding/detection chamber 24, whichincorporates a binding surface which has been modified with a specificbinding reagent comprising antibodies specific for bovine leukocyte cellsurface antigens. The platform also comprises an overflow reservoir 23and waste receptacle 25. The milk sample is introduced, optionally afterpretreatment, for example, to remove fat globules or other non-specificparticulates, into the sample entry port on the platform. Platforms ofthe invention can be provided with wash buffer and stain or can be addedto the platform immediately before use. The platform is rotated at agradually increasing rate to move the excess fluid from the samplechamber into the overflow chamber. The speed is increased further todrive the sample into the binding/detection chamber, where it contactsthe surface coated with the specific binding reagent. The milk is thenincubated in the chamber for 30 minutes. Following incubation, a valveconnecting the wash buffer reservoir to the binding/detection chamber isopened and fluid flow achieved, e.g., by increasing the rotation rate orby actuating a thermal valve, so that the wash buffer flushes the milksample out of the chamber and into the waste receptacle. After the washbuffer has replaced the milk in the binding/detection chamber, a valveconnecting the dye chamber containing the staining solution to thebinding/detection chamber is opened so that the staining solution fillsthe binding chamber and replaces the wash buffer. After allowing asufficient time for the dye to stain the cells, the number of cellsbound to the chamber are observed visually using source light atwavelengths between 510 and 560 nm derived by filtering the light of amercury arc lamp, and a long pass filter to detect the emittedfluorescence.

Alternatively, in a mechanically-simpler embodiment, the milk sample ispretreated by adding buffer and dye (described in greater detail in S.Williams (ed.), 1984, Official Methods of Analysis of the AOAC, 14^(th)ed., Assoc. of Official Analytical Chemists, Arlington, Va.)off-platform, then the sample is introduced onto the platform. Theplatform is rotated to transfer the sample into the binding chamberwhere the cells settle and bind to the specific binding reagent. Awashing step is performed by increasing the rotation rate of theplatform to transfer a washing fluid into the binding chamber, followedby cell staining and detection as described above.

EXAMPLE 3 Drug Discovery

Another example of the apparatus and methods of the invention isautomated evaluation of the effect of test molecules on a population ofcells. Advantageously, the cells are bacterial or mammalian cells, andoriginate from either primary cultures (i.e. freshly harvested,particularly hematopoietic cells) or established cell lines. Viability,metabolic activation (as indicated by changes in membrane potential,intracellular pH and/or intracellular free calcium concentration) andother cellular responses are assessed using the detection means of theinvention described here. Test molecules include drugs such asantibiotics, compounds being assessed for cellular toxicity, ormolecules being screened to activate or block a particular receptor on acell. The platforms or disks of the invention are used generallyaccording to the invention as follows. Cells are cultured or isolated,stained with a suitable dye, exposed to the test molecule, and the cellsthen analyzed by monitoring or measuring the optical properties of thecells associated with cell viability, metabolic activation or othercellular response to the test compound. The sequence in which theseoperations are performed is dependent on the test compound; in certainembodiments these operations are performed sequentially while in othersthe operations are performed simultaneously.

One example of a preferred method of toxicology testing useful with theplatforms of the invention is the neutral red uptake test (A. M.Goldberg and J. M. Frazier, 1989, “Alternatives to Animals in ToxicityTesting”, Sci. Amer. 261: 24-30). In this assay, cells are exposed to acytological dye (specifically, a vital stain) and a potential toxin, andthe viability of the cells detected after incubation is related directlyto the amount of dye taken up by the cells. In another application, drugdiscovery, cells are stained with fluorescent indicators that respond tochanges in membrane potential, intracellular pH or ion concentrations,and then challenged with the test molecule. The cytological response ismeasured using an optical detection system which detects fluorescence.In a particular application in the field of drug discovery,high-throughput screening of potentially biologically active compounds,e.g., test molecules that activate a particular receptor on a cell ofinterest are contacted with cells and the binding constant of successfulcandidate test molecules determined by studying the dependence of testmolecule concentration on cell activation. For example, in Type II, oradult-onset, diabetes, treatment consists of administering drugs whichincrease insulin production by stimulating the beta cells of thepancreas. One class of drug administered in this treatment issulfonylureas, which are believed to act by blocking ATP-dependentpotassium ion channels. As a consequence of sulfonylurea treatment, thecells are depolarized, leading to opening of voltage dependent calciumion channels and increased insulin secretion. A platform arrangementuseful in high throughput screening for insulin-stimulating drugs isillustrated in FIG. 2. The platform consists of a cell accumulationchamber 24 in which cells are cultured; a test compound buffer chamber29 for introducing a predetermined concentration of the test compound tothe cell accumulation chamber; a dye chamber containing a cytologicalstain solution 27; a wash buffer chamber 28; and a waste receptaclechamber 25. Rat insulinoma cells (strain RINm5F) are resuspended inculture medium (e.g. RPMI 1640 supplemented with serum, antibiotics andglucose) and introduced onto the accumulation chamber of the device. Thecells are then cultured at 37° C. for a sufficient period of time toallow the cells to attach to the platform surface. Following attachment,the staining and test molecule chambers are loaded. The cells arestained with a solution of a calcium-sensitive fluorescent dye (e.gFluo-3) prepared in a buffered medium (e.g. HEPES-NaOH) supplementedwith glucose at a dye concentration of about 1 to 10 micromolar. Asolution of a test molecule (e.g. 1 mM tolbutamide) in the same bufferis added to the test molecule chamber. The assay is initiated byrotating the platform at a gradually increasing rate with opening of avalve connecting the stain to the cell accumulation chamber, andincreasing the rotation rate or actuating a thermal valve, so that thestain flushes the cell culture medium out of the chamber. The stainingsolution is allowed to remain in the chamber for a sufficient period oftime to effect staining of the cells (from minutes to an hour).Following this, the valve connecting the test molecule to the bindingchamber is opened (by increasing rotation rate or actuating a thermalvalve). The solution of the test molecule solution replaces the stainsolution and fills the cell accumulation chamber. The effect of the testcompound on the cells is assayed by determining intracellular calciumconcentration by detecting fluorescence of the cells using fluorescenceoptics, such as a fluorescence microscope using a filtered (450-490 nm)mercury lamp as light source, and a photomultiplier tube and filter(passing wavelengths greater than 520 nm); typically, intracellularcalcium concentration is initiated monitored continuously over a timecourse of the assay.

It will be recognized that the methods described in this example can beadvantageously adapted to assays for antibiotic susceptibility ofbacterial cells and toxicological testing.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention.

What is claimed is:
 1. A centripetally-motivated apparatus for detectinga particulate in a fluid, the apparatus comprising in combination aplatform, comprising a substrate having a first flat, planar surface anda second flat, planar surface opposite thereto, and an axis of rotation,wherein the first surface defines a detection chamber comprising an areathat is coated with a specific binding reagent that specifically bindsto the particulate to be detected, and further comprises a multiplicityof microchannels embedded within the first planar surface and a sampleinput means, wherein the detection chamber, the sample input means andthe microchannels are connected and in fluid communication, and whereinthe platform further comprises a wash buffer reservoir containing a washbuffer in fluid communication with the detection chamber; and a fluidwaste receptacle in fluid communication with the detection chamberwherein the wash buffer reservoir and the sample input means arepositioned on the first surface of the platform to be closer to the axisof rotation than is the detection chamber, and wherein the fluid wastereceptacle is positioned on the surface of the platform to be fartherfrom the axis of rotation than the detection chamber; the apparatuscomprising the platform in combination with a device, comprising a base,a rotating means, a power supply and user interface and operationscontrolling means, wherein the rotating means is operatively linked tothe platform and in rotational contact therewith wherein a fluid samplecomprising a particulate is moved from the sample input means to thedetection chamber by centripetal force arising from rotational motion ofthe platform for a time and a rotational velocity sufficient to move thefluid through the microchannels, and is incubated thereon for a timesufficient to result in specific binding between the particulate in thefluid sample and the specific binding reagent; and wherein the fluidsample is replaced with the wash buffer and displaced into the fluidwaste receptacle by centripetal force arising from rotational motion ofthe platform for a time and a rotational velocity sufficient to move thefluid through the microchannels; and wherein the wash buffer is furtherdisplaced into the fluid waste receptacle by centripetal force arisingfrom rotational motion of the platform for a time and a rotationalvelocity sufficient to move the fluid through the microchannels; andwherein a particulate specifically bound to the detection chamber isdetected thereupon.
 2. An apparatus according to claim 1 wherein thesurface of the detection chamber is a porous surface.
 3. An apparatus ofclaim 1 wherein the sample input means further comprises a meteringfluid sample application means, wherein a metered amount of an unmeteredfluid sample applied to the sample input means is retained in themetering fluid sample application means, wherein the sample input meansis further in fluidic contact with an overflow reservoir, wherein fluidcomprising the unmetered fluid sample in excess of the metered amount ofthe fluid sample that is retained by the metering sample applicationmeans is transferred to the overflow reservoir.
 4. An apparatusaccording to claim 3 wherein the metered amount of the fluid sample isfrom about 10 μL to about 500 μL.
 5. The apparatus of claim 1, whereinthe first flat, planar surface and second flat, planar surface of theplatform form a disk.
 6. The apparatus of claim 1, wherein the first andsecond flat, planar surfaces of the platform define a centrally locatedaperture that is engaged to a spindle on the device, whereby rotationalmotion of the spindle is translated into rotational motion of theplatform.
 7. An apparatus according to claim 1, wherein the particulateis a cell.
 8. An apparatus according to claim 1, further comprising: areservoir comprising an amount of a detectable labeling moiety that isspecific for the particulate to be detected and is in fluidcommunication with the detection chamber, wherein the wash buffer isdisplaced from the detection chamber by the detectable labeling moiety.9. An apparatus according to claim 8, wherein the detectable labelingmoiety is a histochemical stain.
 10. An apparatus according to claim 9wherein the detectable labeling moiety is a immunochemical reagent. 11.An apparatus according to claim 10 wherein the immunochemical reagent isa detectably labeled antibody.
 12. An apparatus according to claim 11wherein the detectable label is produced by an enzymatic moiety, whereinthe apparatus further comprises a substrate for the enzymatic moietywherein the substrate is converted to a detectable label by enzymaticaction.
 13. An apparatus according to claim 12 wherein the substratecomprises a component of the wash buffer.
 14. An apparatus according toclaim 12 wherein the substrate comprises a component of the reservoircontaining the detectable labeling moiety.
 15. An apparatus according toclaim 8 wherein the detectable label is a fluorescent label.
 16. Anapparatus according to claim 1 wherein the wash buffer further comprisesa detectable label.
 17. An apparatus according to claim 1 wherein thedetection chamber comprises a volume from about 5 μL to about 1000 μL.18. An apparatus according to claim 1 wherein the device furthercomprises a light source positioned to illuminate the platform.
 19. Anapparatus according to claim 18 further comprising a photodetector,wherein the photodetector is positioned to detect light from the lightsource transmitted through or reflected from the detection chamber. 20.The apparatus of claim 19, wherein the photodetector is brought intoalignment with the detection chamber by rotational motion of theplatform.
 21. The apparatus of claim 18, wherein the photodetector isstationary and samples the detection chamber at a frequency equal to thefrequency of rotation of the platform or multiples thereof.
 22. Anapparatus according to claim 18 wherein a portion of the detectionchamber is optically transparent, and the photodetector and the lightsource are positioned so that light from the light source illuminatesthe detection chamber and is detected by the photodetector through theoptically transparent portion of the platform.
 23. An apparatusaccording to claim 18 wherein a portion of the detection chambercomprises a reflective surface, and the photodetector and the lightsource are positioned so that light from the light source illuminatesthe detection chamber, is reflected therefrom and the reflected light isdetected by the photodetector.
 24. An apparatus according to claim 18wherein the detection chamber further comprises alternating transparentand reflective regions.
 25. An apparatus according to claim 24, whereinthe alternating transparent and reflective regions define a pattern inthe area.
 26. An apparatus according to claim 24 wherein the specificbinding reagent is present in the detection chamber on the surface ofthe platform on a transparent region thereof.
 27. An apparatus accordingto claim 1 wherein the detection chamber is further treated with ablocking agent that prevents non-specific binding to the surface of theplatform.
 28. An apparatus according to claim 27 wherein the specificbinding reagent is present in the detection chamber on the surface ofthe platform on transparent regions thereof, and the blocking reagent ispresent in the detection chamber on the surface of the platform ontransparent and reflective regions thereof.
 29. A method for detecting acell in a fluid using an apparatus according to claim 1, the methodcomprising the steps of: applying an amount of a sample to the fluidsample input means of the apparatus; moving the fluid sample from thefluid sample input means to the detection chamber; incubating the fluidsample in the detection chamber and replacing the fluid of the fluidsample in the detection chamber with the wash buffer, wherein the cellsare retained in the detection chamber; displacing the wash buffer intothe fluid waste receptacle, wherein the cells are retained in thedetection chamber; and detecting the particulate retained in thedetection chamber.